Mucociliary function following- sinus mucosal regeneration MICHAEL S. BENNINGER, MD, JONATHAN L. SCHMIDT, MD, JOHN D. CRISSMAN, MD. and CARY GOTTLIEB, MD, Detroit, Michigan
To evaluate sinus mucosal regeneration and cilia motility after surgical removal, 15 rabbits underwent unilateral removal of maxillary sinus mucosa. The opposite sinus served as an unoperated control. After10 weeks, specimens were taken for examination from all operated on and control sinuses. light, dark-field, and electron microscopy were performed. Mucosa from eight of the 15 sinuses operated on showed ciliary regeneration by light microscopy; six of these exhibited motile cilia on dark-field examination. Histopathologic findings of marked fibrosis, decreased seromucinous glands, and significant inflammation were commonly present in the sinuses operated on. Electron microscopy revealed frequent abnormalities, including complex and edematous cilia and fewer than normal cilia per unit area compared to controls. The dark-field, light, and electron microscopic findings of the regenerated, post-surgical mUCOSa are discussed. (OTOLARYNGOL HEAD NECK SURG 1991;105:641,)
T h e sinus mucociliary transport mechanism is dependent on a complex interaction of cilia motility; the characteristics of the mucus, glandular secretion, and absorption; and the sinus-nasal environment. Chronic inflammatory diseases of the nose and paranasal sinuses can inhibit mucociliary transport, resulting in mucus stasis and secondary sinusitis. Surgical removal of sinus mucosa for such disorders raises questions concerning mucosal regeneration and subsequent sinus
shown that mucociliary clearance was observable in 46% of rabbit sinuses after mucosal removal, but only 15% followed a normal pattern of transport toward and through the natural o ~ t i u m . ~ The purposes of this study were to evaluate the regeneration of maxillary sinus mucosa, the ciliary activity, and the cilia ultrastructure after complete surgical removal of maxillary sinus mucosa. METHODS AND MATERIALS
In an earlier study,4 it was found that in rabbits, after a Caldwell-Luc procedure with complete removal of sinus mucosa, regenerated maxillary sinus mucosa had increased acute and chronic inflammation, granulation tissue, and fibrosis in comparison to control unoperated sinuses. Although ciliated respiratory epithelium was found in some of the sinuses previously operated on, no conclusions could be made as to the function of the mucociliary transport system. A subsequent study has
From the Departments of Otolaryngology-Head and Neck Surgery (Drs. Benninger and Schmidt) and Pathology (Drs. Crissman and Gottlieb), Henry Ford Hospital. Presented at the European Rhinologic Society Meeting. 13th Congress, London, England, June 24-29, 1990. Received for publication Aug. 1, 1990: revision received March 1 I , 1991; accepted April 25, 1991. Reprint requests: Michael S. Benninger, MD, Department of Otolaryngology-Head and Neck Surgery, Henry Ford Hospital, 2799 West Grand Blvd., Detroit, MI 48202-2689. 23/ 1 /30656
Fifteen Pasteurella-free New Zealand white rabbits, weighing 2.4 to 2.8 kg each, were anesthetized with intramuscular injections of ketamine hydrochloride (40 to 50 mg/kg) and Xylozine (7.5 mg/kg). The areas over the bridge of the nose and maxillary sinuses were shaved and then prepared with povidone-iodine (Betadine). The bridge of the nose was infiltrated with 1 % lidocaine (Xylocaine) with 1 : 200,000 epinephrine before an incision was made in the midline of the nose through the skin and periosteum. The periosteum was then elevated and stripped over the maxillary sinus. Alternate sides served as the sinus operated on, with the opposite side becoming the unoperated control. The anterior wall of the sinus was entered and the sinus mucosa was stripped with curettes and small biting forceps. A small antrostomy was then made into the nose, inferior to the natural ostia, in an area previously shown to be effective in preventing suppurative sinusitis.' The skin was then closed with chromic sutures. Bacitracin ointment was applied to the incision postoperatively and for the following 5 days. The rabbits received tetra641
642 BENNINGER et 01.
OtolaryngologyHead and Neck Surgery
Fig. I.A, Normal sinus mucosa from non-operatedsinus. Note the pseudostratifiedciliated columnar epithelium and seromucinousglands. B, Mucosa from operated sinus. There is an irregular epithelium present, which is only focally ciliated. The seromucinous glands have largely been replaced by a chronic inflammatoty (lymphocytic) infiltrate with some fibrosis (Hematoxylin-eosin;original magnification x 100.)
cycline, added to their drinking water for 2 days preoperatively and 5 days postoperatively. After allowing 10 weeks for healing and regrowth of epithelium, the animals were reanesthetized and the
incisions were reopened. The anterior walls of both sinuses were entered and specimens were taken from each sinus for biopsy and placed in normal saline. The animals were then killed.
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Mucociliary function following sinus mucosal regeneration 643
B
Fig. 2. A, Electron microscopic view of non-operatedsinus mucosa. Note the density of cilia and lack of abnormal ciliary structures. B, Operated sinus mucosal cell. There is a dramatic decrease in cilia present (Original magnification x 13,600.)
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644 EENNINGER et a1
A
5;.
Fig. 3. A and 6, Ultrastructural features of abnormal cilia can be appreciated in the mucoso from the operative site. There are edematous blebs (ma// arrows], compound cilia (large arrows]. ond abnormalities of the normal 9 + 2 ciliary architecture (38. curved arrow] (Original magnification: A, x 81,000; B, x 22,800.)
Volume 105 Number 5 November 1991
Mucociliary function following sinus mucosal regeneration 645
Fig. 4. Electron microscopic view of operated sinus mucosa.Lines drawn perpendicularto the central ciliary doublet reveal that the cilia are oriented within a 30-degree range, which is within normal limits [Original magnification: x 20,400.)
The specimens were transported in saline and were then randomly divided into portions for viability testing. determination of cilia movement. routine histology, and electron microscopy. In order to assess whether absence of cilia motility was a result of cell nonviability. a trypan blue dye exclusion test was performed. A drop of dye was added to fresh saline-immersed tissue fragment and observed microscopically for niucosal cell dye exclusion (viable) or uptake (nonviable). The presence of ciliary movement was determined by viewing fresh saline-immersed tissue with dark-field niicroscopy. Routine heniatoxylin-eosin stained slides were prepared from formalin-fixed (405%volume / volume) paraffin-embedded tissue for all specimens from both operated and control sinuses. Specimen blocks were rotated 90" and recut. preventing orientation difficulties encountered with niucosal viability and cilia motility testing. For electron microscopy. small fragments were minced into I-mni slices, put into cold 3.5% glutaraldehyde. rinsed in buffer. post-fixed in I %- osniic acid, dehydrated with alcohol. and embedded in epon. Thin sections were prepared and stained with uranyl acetate and lead citrate, and examined in a Zeiss transmission electron microscope (Carl Zeiss, New York. N.Y.). Photography was done at 50 kv. All specimens were
analyzed by the same pathologist, who was unaware as to whether they represented control sinuses or sinuses operated on. RESULTS Mucosal viability. In one control and five operated on samples. the mucosa was not identified (i.e., sample was predominantly fibrous tissue and glands, lacking a niucosal surface. or was not oriented so that niucosa could be visualized). The remaining 14 eontrol and 10 operated on samples were lined by viable niucosa. Cilia motility. All 15 control and all 15 operated on samples were examined for cilia motility by dark-field microscopy. Six of 15 control and 6 of 15 operated on specimens demonstrated cilia motility. In one operated on sample. cilia movement was focal and weak. The inability to observe cilia motility may have resulted from either lack of ciliated mucosal surfaces or difficulty orienting the mucosa perpendicular to the light source. Routine light microscopy. Of the 15 control samples. cilia were found on 14. and these were associated with a normal pseudostratified columnar mucosa (Fig. I . A ) . A slight decrease in submucosal glands was seen in two of IS. One of 15 had a chronic inflammatory
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646 BENNINGER et al.
Table 1. Summary of non-operated vs. operated pathology findings
Mucosal viability Cilia motility (darkfield) Light microscopy (H + E ) Mucosa and submucosa present Submucosa present/no mucosa Ciliated mucosa present Decreased submucosal glands Increased submucosal inflammation Fibrosis in submucosa Electron microscopy Number of cilia Abnormal cilia (Compound-edematous cilia) (H
Non-operated
Operated
14 of 15 6 o f 15
10 of 15 6 o f 15
15
12 of 15 1 of 15 a of 12 10 of 13 7 0 f 13 13 of 13
15 of 0 14 of 2 of 1 of 4of
15 15
15 15
100 of 100 1%
54 of 100 (54%) 10%
+ €), Hernatoxylin-eosin stain
infiltrate present, whereas four had mild-to-moderate fibrosis present. The samples operated on demonstrated a higher incidence of abnormal findings (Fig. 1, B ) . Eight of the 12 samples with mucosa present had ciliated epithelium. However, five of these eight cases contained hypertrophic mucosa (i.e., elongated epithelial cells), which was not seen in any of the samples not operated on. One of 12 had a nonciliated pseudostratified columnar epithelium present. Three of 12 lacked normal mucosal epithelium, but had a flattened cuboidal epithelium, lacking cilia. Submucosal glands were decreased in 10 of 12 cases that had the submucosal tissue available for study. In one of 12, the glands were slightly increased in number. The histology of the glands was that of unremarkable seromucinous glands. Acute and chronic inflammation was found in 7 of 13 samples. Fibrous scarring was found in 13 of 13 samples. Two samples were not included in the light microscopy analysis because they contained either muscle and nerve or fibroadipose connective tissue, but lacked mucosa and submucosa. One sample contained only submucosa and was not included in the 12 cases for mucosal analysis. Electron microscopy. Two representative controls and four representative operated on samples were examined. In comparison to control mucosa (Fig. 2, A ) , the sites operated on had a decreased density of cilia (Fig. 2, B ) . By evaluating the number of ciliary roots per area of specimen and comparing operated and control specimens at the same level of magnification, 54 cilia were present in the specimen operated on for 100 cilia in the controls (54%).While compound and edematous cilia were found occasionally in the control mucosal samples, they were found with a much greater
frequency and a greater number of cilia were present within each compound cilium in the operated specimens (Fig. 3, A and B ) . Approximately 10% of the cilia on the sinuses operated on were abnormal, whereas less than 1% of the sinus cilia not operated on were abnormal. Deviations from the normal 9 2 microtubular arrangement were frequently identified in the operated on cilia (Fig. 3, B ) . Both non-operated and operated on cilia had the central microtubule oriented within a 30-degree range, parallel to neighboring cilia and perpendicular to the cell border (Fig. 4). Summary of the pathologic findings (Table 1). Compared to the control mucosal samples not operated on, the samples operated on tended to have a greater frequency of nonciliated mucosa and decreased submucosal glands, associated with inflammation and fibrous scarring. Not only were there fewer samples with ciliated mucosa, but when present, the ciliary population was diminished by nearly half. Ultrastructurally, these cilia were characterized by a tenfold increase in abnormal microtubular defects and variations in morphology. There were no obvious pathologic differences with regard to mucosal viability or detection of cilia motility.
+
DISCUSSION
The integrity of the mucociliary transport system after complete removal of sinus mucosa has long been a subject of debate.4.5Recent work in rabbits has indicated that ciliated respiratory mucosa can regenerate after surgical removal. Acute and chronic inflammation and decreased seromucinous glands are found in a majority of regenerated sinuses in comparison to controls not operated Such changes can result in increased viscosity of the mucus and decreased cilia motility re-
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Mucociliary function following sinus mucosal regeneration 647
sulting in mucus stasis and recurrent infections.’ Although mucociliary transport can occur, usually in the direction of the normal sinus ostia, transport through the ostia occurred in only two of 13 (15%) sinuses previously operated The normal function of the mucociliary transport system involves coordinated functional synchronized cilia and secretory cells.3 Alterations in the normal environment can result in ineffective mucus transport. The frequency of cilia beating can be decreased by mucosal drying, variations from optimal temperature, or pH, certain drugs, smoking, and e x e r c i ~ e . ~ ~ ’ ~ Ultrastructural ciliary defects seen on electron microscopy can be found in congenital conditions such as primary cilia dyskinesia, immotile cilia syndrome, retinitis pigmentosa, and Kartagener’s syndrome, as well as acquired processes such as chronic respiratory tract diseases and some n e o p l a ~ m s Acute .~ viral upper respiratory tract infections in children have been found to result in dysmorphic cilia with microtubular aberrations that can return to normal in a 2-week p e r i ~ d . ~ Quantitative evaluations in healthy subjects, smokers, and those with chronic rhinitis demonstrate that less than 5% of cilia have ultrastructural abnormalities, which do not differ significantly in the frequency between these three groups. ‘ I Marked increases in cilia ultrastructural abnormalities can occur and can affect cilia movement. In Kartagener’s syndrome, there are changes in ciliary orientation, as seen by nonparallel central doublet microtubules. The central microtubules are usually oriented in one direction, with cilia-beating occurring perpendicular to them, and can be used as a marker in determining whether coordinated ciliary unidirectional activity occurs.’’ A marked decrease in the number of dynein arms per cilia, abnormal numbers of microtubules, and incomplete microtubules can also be seen.” Similarly, marked increases in cilia ultrastructural abnormalities occur and correlate with a significant decrease in cilia beat frequency in patients with primary cilia dyskinesia. Ciliary motility has been shown to be weak or absent in almost a third of patients with chronic recurring infections in the upper airways. In half of these, ultrastructural abnormalities of cilia could be demonstrated. Edematous cilia or “blebs ,” microtubular, and dynein arm abnormalities have been noted. l 3 Although low frequencies of ciliary ultrastructural abnormalities can occur in normal subjects, increases in such abnormalities seem to correlate with chronic diseases of the upper airways, where recurrent infections are frequent. In the present study, frequent ciliary
’’
ultrastructural changes such as compound or multiple cilia, when two or more axonemes become enclosed by one cell membrane, and edematous cilia or “blebs” were found in the sinuses operated on. This might be associated with the reparative process or the chronic inflammation present. The total number of cilia present was decreased in the sinuses operated on in comparison to controls. Such abnormalities might result in decreased or absent ciliary beat frequency and decreased or ineffective mucociliary transport. The orientation of the central microtubule pair is parallel in those sinuses operated on that regenerated ciliated mucosa, which might suggest synchronous, directed transport. The decreased number of total cilia and high percentage of abnormal cilia, however, would suggest that in the sinus operated on mucosa fewer than one half of the cilia would be expected to function normally, as compared to the controls. Dark-field microscopy was found to be effective in assessing cilia motility. Only six of the 15 control samples had motility on dark-field examination, but 14 of the 15 specimens had cilia on routine microscopy. Similarly six of the 15 sinuses operated on had motile cilia on dark-field examination, whereas eight of these had cilia on routine microscopy. The false-negative rate would appear to be excessively high. This is probably because of the difficulty orienting the specimen perpendicular to the light in order to visualize movement. This study would be a good predictor of cilia motility if it is present, but would not rule out motile cilia if the test was negative. Furthermore, dark-field examination does not determine if ciliary movement is synchronous and oriented. Its usefulness lies in its use with other studies, such as light and electron microscopy. Glandular secretions and the quality of mucus are also important aspects of the mucociliary transport mechanism. The regenerated mucosa in the sinuses operated on had marked decreased seromucinous glands and increased acute and chronic inflammation, which might be expected to result in increased mucus viscosity and decreased rate of clearance. Radical removal of maxillary sinus mucosa results in contracted sinuses and sinus tissue proliferation, with poor mucociliary clearance toward the natural ~ s t i a .Studies ~ . ~ in rabbits have shown that removal of longitudinal strips of mucosa between the floor of the sinus and the natural ostia, without manipulation of mucosa around the ostia, resulted in decreased rate of mucociliary transport measured by radionucleide scanning in comparison to controls. l4 The present study suggests that complete removal of sinus mucosa can result in poor postoperative sinus
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648 BENNINGER et al.
function. Almost half of the sinuses do not reconstitute with ciliated respiratory mucosa and most have marked inflammation and fibrosis. The postsurgical changes in these sinuses would be expected to prevent normal sinus function. In the sinuses with regenerated mucosa, acute and chronic inflammation and decreased seromucinous glands would be expected to result in mucus reduction and alteration in transport. The ciliary abnormalities noted also may result in decreased ciliary movement with poor mucus blanket transport. The marked decrease in normal functioning cilia might explain why reconstituted mucosa has been found to have functional cilia, but mucociliary clearance through the natural ostia is p00r.~ Although it is difficult to correlate animal studies to human beings, the patterns of mucociliary clearance of the rabbit maxillary sinus is similar to those in human being^.^ Complete surgical removal of the sinus mucosa results in poor regeneration of sinus mucosa with abnormal cilia and glands, as well as chronic inflammatory changes. Absence of mucociliary clearance would be expected in those with no regeneration, and an impediment would be expected in most of those that do regenerate. This may result in chronic mucus stasis and subsequent recurrent or frequent inflammation. A conservation approach to maxillary sinus disease, with selective removal of only diseased mucosa while normal mucosa is preserved rather than completely removed, may result in better postoperative sinus function.
Head and Neck Surgery
REFERENCES 1. Stammberger H. Endoscopic endonasal surgery-concepts
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
CONCLUSION
Regeneration of ciliated maxillary sinus mucosa can occur in rabbits after surgical removal. Because many sinuses do not regenerate ciliated mucosa and those that do have frequent abnormalities at both the structural and ultrastructural levels, normal mucociliary transport is unlikely to occur in most of the sinuses completely denuded of epithelium. Partial or selective removal of mucosa may allow regeneration of more normal mucosa, with improved mucociliary transport and sinus function. We would like to acknowledge Thomas Christopherson for his assistance with electron microscopy, and Ms. Nancy Peshkin and Eddie Burks for their photographic assistance.
12.
13.
14.
in treatment of recurring rhinosinusitis. Part I. Anatomic and pathophysiologic considerations. OTOLARYNCOL HEADNECK SURG 1986;94:143-7. Kennedy DW, Zinreich SJ, Rosenbaum AE, Johns ME. Functional endoscopic sinus surgery: theory and diagnostic evaluation. Arch Otolaryngol 1985;111:576-82. Carson JL, Collier AM, Hu SS. Acquired ciliary defects in nasal epithelium of children with acute viral upper respiratory infections. N Engl J Med 1985;312:463-8. Benninger MS, Sebek BA, Levine HL. Mucosal regeneration of the maxillary sinus after surgery. OTOLARYNGOL HEADNECK SURG 1989; 101:33-7. Kennedy DW, Shaalan H. Reevaluation of maxillary sinus surgery: experimental study in rabbits. Ann Otol Rhinol Laryngol 1989;98:901-6. Hilding A. Experimental surgery of the nose and sinuses. 111. Results following partial and complete removal of the lining mucous membrane from the frontal sinus of the dog. Arch Otolaryngol 1933;17:760-8. Kistner FB. Histopathology and bacteriology of sinusitis with comments on postoperative repair. Arch Otolaryngol 193 1;13:225-37. Hilding AC. Experimental sinus surgery: Effects of operative windows on normal sinuses. Ann Otol Rhinol Laryngol 1941;50:379-92. Taylor M. Physiology of the nose, paranasal sinuses, and nasopharynx. In: English GM, ed. Otolaryngology. Philadelphia: Harper and Row, 1987:1-64. Cederlund A, Camner P, Svartengren M. Nasal mucociliary transport before and after jogging. Phys Sports Med 1987;15: 93-8. Rossman CM, Lee RMKW, Forrest JB, Newhouse MT. Nasal ciliary ultrastructure and function in patients with primary ciliary dyskinesia compared with that in normal subjects and in subjects with various respiratory diseases. Am Rev Respir Dis 1984:!29: I61 -7. Eavey RD, Nadol JB, Holmes LB, et al. Kartagener’s syndrome: a blinded, controlled study of cilia ultrastructure. Arch Otolaryngol Head Neck Surg 1986;112:646-50. Burgersdijk FJA, DeGroot JCMJ, Graamans K, Rademakers LHPM. Testing ciliary activity in patients with chronic and recurrent infections of the upper airways: experiences in 68 cases. Laryngoscope 1986;96:1029-33. Friedman M, Toriumi DM. The effect of a temporary nasoantral window on mucociliary clearance, an experimental study. Otolaryngol Clin North Am 1989;22:819-30.
To evaluate sinus mucosal regeneration and cilia motility after surgical removal, 15 rabbits underwent unilateral removal of maxillary sinus mucosa. The opposite sinus served as an unoperated control. After10 weeks, specimens were taken for examination from all operated on and control sinuses. light, dark-field, and electron microscopy were performed. Mucosa from eight of the 15 sinuses operated on showed ciliary regeneration by light microscopy; six of these exhibited motile cilia on dark-field examination. Histopathologic findings of marked fibrosis, decreased seromucinous glands, and significant inflammation were commonly present in the sinuses operated on. Electron microscopy revealed frequent abnormalities, including complex and edematous cilia and fewer than normal cilia per unit area compared to controls. The dark-field, light, and electron microscopic findings of the regenerated, post-surgical mUCOSa are discussed. (OTOLARYNGOL HEAD NECK SURG 1991;105:641,)
T h e sinus mucociliary transport mechanism is dependent on a complex interaction of cilia motility; the characteristics of the mucus, glandular secretion, and absorption; and the sinus-nasal environment. Chronic inflammatory diseases of the nose and paranasal sinuses can inhibit mucociliary transport, resulting in mucus stasis and secondary sinusitis. Surgical removal of sinus mucosa for such disorders raises questions concerning mucosal regeneration and subsequent sinus
shown that mucociliary clearance was observable in 46% of rabbit sinuses after mucosal removal, but only 15% followed a normal pattern of transport toward and through the natural o ~ t i u m . ~ The purposes of this study were to evaluate the regeneration of maxillary sinus mucosa, the ciliary activity, and the cilia ultrastructure after complete surgical removal of maxillary sinus mucosa. METHODS AND MATERIALS
In an earlier study,4 it was found that in rabbits, after a Caldwell-Luc procedure with complete removal of sinus mucosa, regenerated maxillary sinus mucosa had increased acute and chronic inflammation, granulation tissue, and fibrosis in comparison to control unoperated sinuses. Although ciliated respiratory epithelium was found in some of the sinuses previously operated on, no conclusions could be made as to the function of the mucociliary transport system. A subsequent study has
From the Departments of Otolaryngology-Head and Neck Surgery (Drs. Benninger and Schmidt) and Pathology (Drs. Crissman and Gottlieb), Henry Ford Hospital. Presented at the European Rhinologic Society Meeting. 13th Congress, London, England, June 24-29, 1990. Received for publication Aug. 1, 1990: revision received March 1 I , 1991; accepted April 25, 1991. Reprint requests: Michael S. Benninger, MD, Department of Otolaryngology-Head and Neck Surgery, Henry Ford Hospital, 2799 West Grand Blvd., Detroit, MI 48202-2689. 23/ 1 /30656
Fifteen Pasteurella-free New Zealand white rabbits, weighing 2.4 to 2.8 kg each, were anesthetized with intramuscular injections of ketamine hydrochloride (40 to 50 mg/kg) and Xylozine (7.5 mg/kg). The areas over the bridge of the nose and maxillary sinuses were shaved and then prepared with povidone-iodine (Betadine). The bridge of the nose was infiltrated with 1 % lidocaine (Xylocaine) with 1 : 200,000 epinephrine before an incision was made in the midline of the nose through the skin and periosteum. The periosteum was then elevated and stripped over the maxillary sinus. Alternate sides served as the sinus operated on, with the opposite side becoming the unoperated control. The anterior wall of the sinus was entered and the sinus mucosa was stripped with curettes and small biting forceps. A small antrostomy was then made into the nose, inferior to the natural ostia, in an area previously shown to be effective in preventing suppurative sinusitis.' The skin was then closed with chromic sutures. Bacitracin ointment was applied to the incision postoperatively and for the following 5 days. The rabbits received tetra641
642 BENNINGER et 01.
OtolaryngologyHead and Neck Surgery
Fig. I.A, Normal sinus mucosa from non-operatedsinus. Note the pseudostratifiedciliated columnar epithelium and seromucinousglands. B, Mucosa from operated sinus. There is an irregular epithelium present, which is only focally ciliated. The seromucinous glands have largely been replaced by a chronic inflammatoty (lymphocytic) infiltrate with some fibrosis (Hematoxylin-eosin;original magnification x 100.)
cycline, added to their drinking water for 2 days preoperatively and 5 days postoperatively. After allowing 10 weeks for healing and regrowth of epithelium, the animals were reanesthetized and the
incisions were reopened. The anterior walls of both sinuses were entered and specimens were taken from each sinus for biopsy and placed in normal saline. The animals were then killed.
Volume 105 Number 5 November 1991
Mucociliary function following sinus mucosal regeneration 643
B
Fig. 2. A, Electron microscopic view of non-operatedsinus mucosa. Note the density of cilia and lack of abnormal ciliary structures. B, Operated sinus mucosal cell. There is a dramatic decrease in cilia present (Original magnification x 13,600.)
OtolaryngologyHead and Neck Surgery
644 EENNINGER et a1
A
5;.
Fig. 3. A and 6, Ultrastructural features of abnormal cilia can be appreciated in the mucoso from the operative site. There are edematous blebs (ma// arrows], compound cilia (large arrows]. ond abnormalities of the normal 9 + 2 ciliary architecture (38. curved arrow] (Original magnification: A, x 81,000; B, x 22,800.)
Volume 105 Number 5 November 1991
Mucociliary function following sinus mucosal regeneration 645
Fig. 4. Electron microscopic view of operated sinus mucosa.Lines drawn perpendicularto the central ciliary doublet reveal that the cilia are oriented within a 30-degree range, which is within normal limits [Original magnification: x 20,400.)
The specimens were transported in saline and were then randomly divided into portions for viability testing. determination of cilia movement. routine histology, and electron microscopy. In order to assess whether absence of cilia motility was a result of cell nonviability. a trypan blue dye exclusion test was performed. A drop of dye was added to fresh saline-immersed tissue fragment and observed microscopically for niucosal cell dye exclusion (viable) or uptake (nonviable). The presence of ciliary movement was determined by viewing fresh saline-immersed tissue with dark-field niicroscopy. Routine heniatoxylin-eosin stained slides were prepared from formalin-fixed (405%volume / volume) paraffin-embedded tissue for all specimens from both operated and control sinuses. Specimen blocks were rotated 90" and recut. preventing orientation difficulties encountered with niucosal viability and cilia motility testing. For electron microscopy. small fragments were minced into I-mni slices, put into cold 3.5% glutaraldehyde. rinsed in buffer. post-fixed in I %- osniic acid, dehydrated with alcohol. and embedded in epon. Thin sections were prepared and stained with uranyl acetate and lead citrate, and examined in a Zeiss transmission electron microscope (Carl Zeiss, New York. N.Y.). Photography was done at 50 kv. All specimens were
analyzed by the same pathologist, who was unaware as to whether they represented control sinuses or sinuses operated on. RESULTS Mucosal viability. In one control and five operated on samples. the mucosa was not identified (i.e., sample was predominantly fibrous tissue and glands, lacking a niucosal surface. or was not oriented so that niucosa could be visualized). The remaining 14 eontrol and 10 operated on samples were lined by viable niucosa. Cilia motility. All 15 control and all 15 operated on samples were examined for cilia motility by dark-field microscopy. Six of 15 control and 6 of 15 operated on specimens demonstrated cilia motility. In one operated on sample. cilia movement was focal and weak. The inability to observe cilia motility may have resulted from either lack of ciliated mucosal surfaces or difficulty orienting the mucosa perpendicular to the light source. Routine light microscopy. Of the 15 control samples. cilia were found on 14. and these were associated with a normal pseudostratified columnar mucosa (Fig. I . A ) . A slight decrease in submucosal glands was seen in two of IS. One of 15 had a chronic inflammatory
OtolaryngologyHead and N e c k Surgery
646 BENNINGER et al.
Table 1. Summary of non-operated vs. operated pathology findings
Mucosal viability Cilia motility (darkfield) Light microscopy (H + E ) Mucosa and submucosa present Submucosa present/no mucosa Ciliated mucosa present Decreased submucosal glands Increased submucosal inflammation Fibrosis in submucosa Electron microscopy Number of cilia Abnormal cilia (Compound-edematous cilia) (H
Non-operated
Operated
14 of 15 6 o f 15
10 of 15 6 o f 15
15
12 of 15 1 of 15 a of 12 10 of 13 7 0 f 13 13 of 13
15 of 0 14 of 2 of 1 of 4of
15 15
15 15
100 of 100 1%
54 of 100 (54%) 10%
+ €), Hernatoxylin-eosin stain
infiltrate present, whereas four had mild-to-moderate fibrosis present. The samples operated on demonstrated a higher incidence of abnormal findings (Fig. 1, B ) . Eight of the 12 samples with mucosa present had ciliated epithelium. However, five of these eight cases contained hypertrophic mucosa (i.e., elongated epithelial cells), which was not seen in any of the samples not operated on. One of 12 had a nonciliated pseudostratified columnar epithelium present. Three of 12 lacked normal mucosal epithelium, but had a flattened cuboidal epithelium, lacking cilia. Submucosal glands were decreased in 10 of 12 cases that had the submucosal tissue available for study. In one of 12, the glands were slightly increased in number. The histology of the glands was that of unremarkable seromucinous glands. Acute and chronic inflammation was found in 7 of 13 samples. Fibrous scarring was found in 13 of 13 samples. Two samples were not included in the light microscopy analysis because they contained either muscle and nerve or fibroadipose connective tissue, but lacked mucosa and submucosa. One sample contained only submucosa and was not included in the 12 cases for mucosal analysis. Electron microscopy. Two representative controls and four representative operated on samples were examined. In comparison to control mucosa (Fig. 2, A ) , the sites operated on had a decreased density of cilia (Fig. 2, B ) . By evaluating the number of ciliary roots per area of specimen and comparing operated and control specimens at the same level of magnification, 54 cilia were present in the specimen operated on for 100 cilia in the controls (54%).While compound and edematous cilia were found occasionally in the control mucosal samples, they were found with a much greater
frequency and a greater number of cilia were present within each compound cilium in the operated specimens (Fig. 3, A and B ) . Approximately 10% of the cilia on the sinuses operated on were abnormal, whereas less than 1% of the sinus cilia not operated on were abnormal. Deviations from the normal 9 2 microtubular arrangement were frequently identified in the operated on cilia (Fig. 3, B ) . Both non-operated and operated on cilia had the central microtubule oriented within a 30-degree range, parallel to neighboring cilia and perpendicular to the cell border (Fig. 4). Summary of the pathologic findings (Table 1). Compared to the control mucosal samples not operated on, the samples operated on tended to have a greater frequency of nonciliated mucosa and decreased submucosal glands, associated with inflammation and fibrous scarring. Not only were there fewer samples with ciliated mucosa, but when present, the ciliary population was diminished by nearly half. Ultrastructurally, these cilia were characterized by a tenfold increase in abnormal microtubular defects and variations in morphology. There were no obvious pathologic differences with regard to mucosal viability or detection of cilia motility.
+
DISCUSSION
The integrity of the mucociliary transport system after complete removal of sinus mucosa has long been a subject of debate.4.5Recent work in rabbits has indicated that ciliated respiratory mucosa can regenerate after surgical removal. Acute and chronic inflammation and decreased seromucinous glands are found in a majority of regenerated sinuses in comparison to controls not operated Such changes can result in increased viscosity of the mucus and decreased cilia motility re-
Volume 105 Number 5 November 1991
Mucociliary function following sinus mucosal regeneration 647
sulting in mucus stasis and recurrent infections.’ Although mucociliary transport can occur, usually in the direction of the normal sinus ostia, transport through the ostia occurred in only two of 13 (15%) sinuses previously operated The normal function of the mucociliary transport system involves coordinated functional synchronized cilia and secretory cells.3 Alterations in the normal environment can result in ineffective mucus transport. The frequency of cilia beating can be decreased by mucosal drying, variations from optimal temperature, or pH, certain drugs, smoking, and e x e r c i ~ e . ~ ~ ’ ~ Ultrastructural ciliary defects seen on electron microscopy can be found in congenital conditions such as primary cilia dyskinesia, immotile cilia syndrome, retinitis pigmentosa, and Kartagener’s syndrome, as well as acquired processes such as chronic respiratory tract diseases and some n e o p l a ~ m s Acute .~ viral upper respiratory tract infections in children have been found to result in dysmorphic cilia with microtubular aberrations that can return to normal in a 2-week p e r i ~ d . ~ Quantitative evaluations in healthy subjects, smokers, and those with chronic rhinitis demonstrate that less than 5% of cilia have ultrastructural abnormalities, which do not differ significantly in the frequency between these three groups. ‘ I Marked increases in cilia ultrastructural abnormalities can occur and can affect cilia movement. In Kartagener’s syndrome, there are changes in ciliary orientation, as seen by nonparallel central doublet microtubules. The central microtubules are usually oriented in one direction, with cilia-beating occurring perpendicular to them, and can be used as a marker in determining whether coordinated ciliary unidirectional activity occurs.’’ A marked decrease in the number of dynein arms per cilia, abnormal numbers of microtubules, and incomplete microtubules can also be seen.” Similarly, marked increases in cilia ultrastructural abnormalities occur and correlate with a significant decrease in cilia beat frequency in patients with primary cilia dyskinesia. Ciliary motility has been shown to be weak or absent in almost a third of patients with chronic recurring infections in the upper airways. In half of these, ultrastructural abnormalities of cilia could be demonstrated. Edematous cilia or “blebs ,” microtubular, and dynein arm abnormalities have been noted. l 3 Although low frequencies of ciliary ultrastructural abnormalities can occur in normal subjects, increases in such abnormalities seem to correlate with chronic diseases of the upper airways, where recurrent infections are frequent. In the present study, frequent ciliary
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ultrastructural changes such as compound or multiple cilia, when two or more axonemes become enclosed by one cell membrane, and edematous cilia or “blebs” were found in the sinuses operated on. This might be associated with the reparative process or the chronic inflammation present. The total number of cilia present was decreased in the sinuses operated on in comparison to controls. Such abnormalities might result in decreased or absent ciliary beat frequency and decreased or ineffective mucociliary transport. The orientation of the central microtubule pair is parallel in those sinuses operated on that regenerated ciliated mucosa, which might suggest synchronous, directed transport. The decreased number of total cilia and high percentage of abnormal cilia, however, would suggest that in the sinus operated on mucosa fewer than one half of the cilia would be expected to function normally, as compared to the controls. Dark-field microscopy was found to be effective in assessing cilia motility. Only six of the 15 control samples had motility on dark-field examination, but 14 of the 15 specimens had cilia on routine microscopy. Similarly six of the 15 sinuses operated on had motile cilia on dark-field examination, whereas eight of these had cilia on routine microscopy. The false-negative rate would appear to be excessively high. This is probably because of the difficulty orienting the specimen perpendicular to the light in order to visualize movement. This study would be a good predictor of cilia motility if it is present, but would not rule out motile cilia if the test was negative. Furthermore, dark-field examination does not determine if ciliary movement is synchronous and oriented. Its usefulness lies in its use with other studies, such as light and electron microscopy. Glandular secretions and the quality of mucus are also important aspects of the mucociliary transport mechanism. The regenerated mucosa in the sinuses operated on had marked decreased seromucinous glands and increased acute and chronic inflammation, which might be expected to result in increased mucus viscosity and decreased rate of clearance. Radical removal of maxillary sinus mucosa results in contracted sinuses and sinus tissue proliferation, with poor mucociliary clearance toward the natural ~ s t i a .Studies ~ . ~ in rabbits have shown that removal of longitudinal strips of mucosa between the floor of the sinus and the natural ostia, without manipulation of mucosa around the ostia, resulted in decreased rate of mucociliary transport measured by radionucleide scanning in comparison to controls. l4 The present study suggests that complete removal of sinus mucosa can result in poor postoperative sinus
Otolaryngology-
648 BENNINGER et al.
function. Almost half of the sinuses do not reconstitute with ciliated respiratory mucosa and most have marked inflammation and fibrosis. The postsurgical changes in these sinuses would be expected to prevent normal sinus function. In the sinuses with regenerated mucosa, acute and chronic inflammation and decreased seromucinous glands would be expected to result in mucus reduction and alteration in transport. The ciliary abnormalities noted also may result in decreased ciliary movement with poor mucus blanket transport. The marked decrease in normal functioning cilia might explain why reconstituted mucosa has been found to have functional cilia, but mucociliary clearance through the natural ostia is p00r.~ Although it is difficult to correlate animal studies to human beings, the patterns of mucociliary clearance of the rabbit maxillary sinus is similar to those in human being^.^ Complete surgical removal of the sinus mucosa results in poor regeneration of sinus mucosa with abnormal cilia and glands, as well as chronic inflammatory changes. Absence of mucociliary clearance would be expected in those with no regeneration, and an impediment would be expected in most of those that do regenerate. This may result in chronic mucus stasis and subsequent recurrent or frequent inflammation. A conservation approach to maxillary sinus disease, with selective removal of only diseased mucosa while normal mucosa is preserved rather than completely removed, may result in better postoperative sinus function.
Head and Neck Surgery
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CONCLUSION
Regeneration of ciliated maxillary sinus mucosa can occur in rabbits after surgical removal. Because many sinuses do not regenerate ciliated mucosa and those that do have frequent abnormalities at both the structural and ultrastructural levels, normal mucociliary transport is unlikely to occur in most of the sinuses completely denuded of epithelium. Partial or selective removal of mucosa may allow regeneration of more normal mucosa, with improved mucociliary transport and sinus function. We would like to acknowledge Thomas Christopherson for his assistance with electron microscopy, and Ms. Nancy Peshkin and Eddie Burks for their photographic assistance.
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