Chem. Senses 28: 33-43,
2003
© Oxford University Press 2003
The Time Course of Taste Bud Regeneration after Glossopharyngeal or Greater Superficial Petrosal Nerve Transection in Rats
1 Department of Psychology, Reed College, Portland OR 97202, USA 2 Department of Psychology, Gainesville, FL 32608, USA
Correspondence to be sent to: Steven St. John, Department of Psychology, Reed College, 3203 SE Woodstock Blvd, Portland, OR 97202. e-mail Steven.St.John{at}reed.edu
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Abstract |
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We previously have published data detailing the time course of taste bud regeneration in the anterior tongue following transection of the chorda tympani (CT) nerve in the rat. This study extends the prior work by determining the time course of taste bud regeneration in the vallate papilla, soft palate and nasoincisor ducts (NID) following transection of either the glossopharyngeal (GL) or greater superficial petrosal (GSP) nerve. Following GL transection in rats (n = 6 per time point), taste buds reappeared in the vallate papilla between 15 and 28 days after surgery, and returned to 80.3% of control levels (n = 12) of taste buds by 70 days postsurgery. The first appearance and the final percentage of the normal complement of regenerated vallate taste buds after GL transection resembled that seen previously in the anterior tongue after CT transection. However, in the latter case, regenerated taste buds reached asymptotic levels by 42 days after surgery, whereas within the time frame of the present study, a clear asymptotic return of vallate taste buds was not observed. In contrast to the posterior (and anterior) tongue, only 25% of the normal complement of palatal taste buds regenerated by 112 days and 224 days after GSP transection (n = 9). The difference in regenerative capacity might relate to the surgical approach used to transect the GSP. These experiments provide useful parametric data for investigators studying the functional consequences of gustatory nerve transection and regeneration.
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Introduction |
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Taste buds are distributed in several distinct receptor fields in the oral cavity of the rat (Miller, 1977
). The chorda tympani branch (CT) of cranial nerve VII
innervates the taste buds of the anterior two-thirds of the tongue. These
taste buds are housed in specialized protrusions on the lingual surface called
fungiform papillae. In the rat, each papilla usually houses a single taste bud
(Miller and Preslar, 1975).
The lingual—tonsilar branch of the glossopharyngeal nerve (GL; cranial
nerve IX) innervates the taste buds of the posterior tongue. These taste buds
are found primarily in the vallate and foliate papillae. In the rat, the
vallate papilla is a single crescent-shaped trench positioned along the
midline of the posterior dorsal surface of the tongue. The foliate papillae
are a series of several trenches on the lateral margins of the posterior
tongue. Taste buds in the vallate and foliate papillae are found lining the
walls of the trenches. A few taste buds in the anterior trench of the foliate
are innervated by the CT.
The palatal taste buds are distributed in three fields. The most anterior
field is found in the incisive papilla associated with the nasoincisor ducts
(NID) in the hard palate just behind the upper incisors. Taste buds can be
found in the medial wall of the ducts near their opening in the oral cavity.
The second field consists of a strip of taste buds at the border of the hard
and soft palates referred to as the `Geschmacksstreifen' or palatal taste
stripe. The third field of taste buds is found in the soft palate proper and
is referred to as the posterior palatine field. Virtually all of the palatal
taste buds are innervated by the greater superficial petrosal branch (GSP) of
cranial nerve VII, but there is evidence that a few may be innervated by
another nerve (Cleaton-Jones,
1976; Miller and Spangler,
1982). The superior laryngeal branch of cranial nerve X innervates
the taste buds of the laryngeal epithelium. The exact numbers and proportions
of taste buds in the various fields vary across subjects, species and studies
(Miller, 1977;
Miller and Smith, 1984;
Travers and Nicklas,
1990).
In the rat, as is the case in other species, the taste buds are trophically
dependent on the innervating nerve (von
Vintschgau and Honigschmied, 1876;
Whiteside, 1927;
Guth, 1957;
Cleaton-Jones, 1976;
Cheal and Oakley, 1977;
Miller, 1977;
Miller and Spangler, 1982;
Ganchrow and Ganchrow, 1989a;
Hard af Segerstad et al.,
1989; Barry and Frank,
1992; Oakley et al.,
1993; St. John et
al., 1995; Ninomiya,
1998). Thus when gustatory nerves are transected, the taste buds
in their respective receptor fields degenerate. One partial exception to this
rule, which is more noteworthy in the hamster than in the rat, involves the
taste buds of the anterior tongue, some of which seem to degenerate
incompletely following resection of the CT
(Whitehead et al.,
1987; Hard af Segerstad et
al., 1989; Oakley et al.,
1990,
1993;
Parks and Whitehead, 1998;
St. John et al.,
1995). However, even in this receptor field, these buds are
readily discriminated in cross-section by their smaller size and atrophic
appearance; in addition, surface stains like methylene blue readily
discriminate innervated and denervated buds because denervation reliably
alters the structure of the taste pore in taste buds of the fungiform papillae
(Parks and Whitehead,
1998).
When damaged or transected in rats, the CT and GL readily regenerate to
reinnervate their appropriate receptor fields causing the regeneration of
taste buds. Regenerated nerves display relatively normal electrophysiological
response profiles to sapid stimuli placed on the tongue
(Cain et al., 1996;
Cheal et al., 1977).
There is also no evidence of intact gustatory nerve fibers sprouting to invade
denervated taste receptor fields normally supplied by a different nerve.
Therefore, the reappearance of taste buds or taste pores following gustatory
nerve transection can be taken as evidence for reinnervation by the transected
nerve (Cheal and Oakley,
1977).
In previous work in rats, we have shown that following bilateral CT
transection in the middle ear, taste buds begin to reappear on the anterior
tongue between 14 and 28 days and reach an asymptotic 
70% return at 42
days. In an exhaustive series of parametric studies of the development and
regeneration of the rat vallate papillae conducted by Bruce Oakley and
colleagues, the time course of unilateral regeneration of a
crushed GL was documented; the contralateral GL was permanently
transected (Hosley and Oakley,
1987; Hosley et al.,
1987a,b;
Oakley, 1993). We are unaware
of any published reports on the time course of regeneration of the GSP or the
superior laryngeal branch of X.
We sought to extend the prior work on the time course of GL regeneration by
examining the return of taste buds after bilateral GL transection.
The taste buds of the vallate papilla receive bilateral innervation from the
GL. Moreover, most studies of the behavioral effects of GL transection involve
bilateral manipulations. A second purpose of this study was to detail the
regeneration of palatal taste buds after GSP transection. Collectively, this
information is potentially useful not only for investigation of regeneration
phenomena, but also for examination of the functional consequences of
gustatory nerve transection. The rapidity with which gustatory nerves can
regenerate in rodents [for example, the CT
(Cheal and Oakley, 1977;
Hard af Segerstad et al.,
1989; St. John et
al., 1995)] sets a limit on the duration of postsurgical
assessment in nerve-transected animals, an approach that has yielded a great
deal of information about the organization of the peripheral gustatory system.
Time-course data allow the investigator to design postsurgical assessments of
gustatory function before the regeneration of the lost taste buds. In other
instances, it may be desirable to relate behavioral performance with the
number of returning taste buds (St. John
et al., 1995). Portions of this work have appeared in
abstract form (St. John et al.,
2002).
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Materials and methods |
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Subjects
Seventy-nine, adult, male Sprague—Dawley rats (CD stock; Charles River Laboratories, Wilmington, MA) served as subjects. The rats were housed individually in hanging, wire mesh cages where food (Laboratory Chow 5001; Purina Mills Inc., St Louis, MO) and tap water were available ad libitum. Temperature, humidity and lighting (12:12 h light—dark cycle) were controlled automatically; all surgical manipulations were performed during the lights-on phase. Each subject participated in only one of two experiments designed to measure the time course of GL regeneration (Experiment 1) or the time course of GSP regeneration (Experiment 2). Subjects were maintained in NIH-approved housing and all procedures were approved by the Institutional Animal Care and Use Committee of the University of Florida.
Experiment 1
Forty-two rats (336-603 g at the start of the experiment) were divided into seven experimental groups (n = 6 per group). Rats in five groups received bilateral transection of the GL (GLX) and rats in the other two groups served as surgical controls (CON). Subjects were anesthetized with a mixture of ketamine hydrochloride (86 mg/kg body mass, i.p.) and xylazine hydrochloride (13 mg/kg body mass, i.p.). All rats also received a prophylactic injection of 30 000 U penicillin (i.m.) on the day prior to surgery.
Rats were placed supine in a customized headholder and an incision was made
in the ventral skin of the neck along the midline. The GL was visualized close
to its exit from the posterior lacerated foramen in the auditory bulla and
inferior to the hypoglossal nerve following retraction of the sublingual and
submaxillary salivary glands, the sternohyoid, omohyoid, and posterior belly
of the digastric muscles. For GLX, the GL was dissected free of the
surrounding fascia, held with a no. 7 microforceps, and cut with
microscissors. The nerve was not damaged beyond this clean cut, and no
explicit attempt was made to approximate the cut ends of the nerve or to form
an anastomosis of the cut ends. For CON, the nerve was visualized but was left
undisturbed. The incision was sutured closed. Because the CON rats also served
as controls in a previously published study of CT regeneration
(St. John et al.,
1995), these rats also had the tympanic membrane punctured with a
no. 7 microforceps. This procedure did not involve removing the rat from the
same headholder; by merely repositioning the rat, the ear canal could be
widened with five blunted and curved hypodermic needles for visualization of
the tympanic membrane.
Rats of the GLX groups were perfused in five groups (14, 28, 42, 56 or 70 days after surgery) and surgical controls in two groups (14 or 70 days). Body weight was monitored after surgery. In two cases, rats were treated with additional penicillin during the survival period. One of these rats also received a liquid nutritional supplement (Precision Diet, Research Diets Inc., New Brunswick, NJ) for 8 days to promote feeding.
For tissue collection, the rats were deeply anesthetized with sodium pentobarbital and perfused transcardially with isotonic saline followed by 10% buffered formalin. The tongue was removed carefully and stored in formalin for at least 5 days. The vallate papilla was embedded in paraffin and sectioned on a rotary microtome (10 µm). The sections were mounted on glass slides and stained with hematoxylin and eosin and the number of taste buds was quantified throughout the entirety of the vallate papilla (see below).
Experiment 2
Thirty-seven rats (318-470 g at the start of the experiment) were divided into nine experimental groups (14 day surgical controls, n = 3; 224 day surgical controls, n = 5; 224 day GSP transection, n = 5; all other groups, n = 4). Rats in six of the groups received bilateral transections of the GSP in the middle ear (GSPX); using this surgical approach the CT was usually also sectioned. In some cases, an attempt to spare the CT was made, but these experiments are not the primary focus of this report. Rats in the remaining three groups served as surgical controls (CON).
Subjects were anesthetized with a mixture of ketamine hydrochloride (125 mg/kg body mass, i.m.) and xylazine hydrochloride (5 mg/kg body mass, i.m.), and were treated with 30 000 U penicillin (s.c.) the day of and for 3 days after surgery. The modification of our anesthetic protocol was motivated by intervening observations that i.m. injection of ketamine—xylazine was a safer alternative to achieving sustained surgical levels of anesthesia in Sprague—Dawley rats.
The rats were then fixed in a custom headholder with the head tilted 80° away from the surgeon. For GSPX, an incision was made around the external ear and the pinna retracted. The ear canal was then punctured and widened by careful dissection of the fascia and retraction of the surrounding musculature. The bony meatus was enlarged with a drill. The tympanic membrane, ossicles, tensor tympani muscle, and a small piece of temporal bone were removed to expose the GSP, which was cut with microforceps. In general it was expected that the distal and proximal ends of the cut nerve remained in approximation, but no explicit attempt was made to form an anastomosis of the cut ends of the nerve. The incision was closed with wound clips. For CON, the pinna was retracted and the soft tissue of the ear canal was widened as described above, and the tympanic membrane punctured, but the internal structures of the middle ear were undisturbed.
For 3 days after surgery, rats were offered a highly palatable,
high-calorie diet of Purina 5001 powder chow mixed with 2.5 g Nutrical
(IGI EVSCO Pharmaceutical, Buena, NJ) and a sweetened condensed milk diet
(diluted 50% with vitamins added). The rats were supplied with wet mash until
body weight was restored to the presurgical value. Rats after GSPX are often
hypophagic for weeks (St. John et
al., 1994), and thus, several different procedures were used
to maintain the health of these rats in the postoperative interval. Milk diet
was continued for some rats beyond the first 3 days, and for others,
occasional injections of penicillin were administered when the possibility of
an infection was suspected.
Rats of the GSPX groups were perfused (as described in Experiment 1) at six different time periods after nerve transection (14, 28, 42, 56, 112 or 224 days after surgery) and CON rats at three different time periods after sham surgery (14, 56 or 224 days after surgery). The soft palate and incisive papilla surrounding NID were removed and stored in 10% buffered formalin. They were later embedded in paraffin and cut, mounted and stained (as described in Experiment 1 for the vallate papilla).
Quantification of taste bud number
In innervated intact taste buds, the apical portions of the taste receptor
cells converge and then protrude into the taste pore; this morphology is not
observed in the atrophic or remnant taste buds sometimes observed in
denervated tissue (Oakley et al.,
1993). This convergence is generally recognizable in just one 10
µm section and is usually accompanied by visualization of a taste pore. Any
taste bud displaying this structural characteristic was counted as
morphologically intact. This counting method helped minimize the possibility
that a single bud would be counted twice. The number of intact taste buds was
quantified for each rat in the relevant taste bud receptor fields.
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Results |
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Experiment 1
The mean number of taste buds was somewhat higher in the CON/70 day than in the CON/14 day (473.83 versus 422.83) group, but this difference was not statistically significant [t(10) = -2.099, P = 0.062]. The groups were combined in subsequent analyses.
Taste buds in the vallate papillae returned as a near-linear function of time since GL transection (Figures 1 and 2). In fact, a linear model accounted for 98.4% of the variance, with a slope of 6.33 taste buds/day. No taste buds were seen at 14 days postsurgery, but if a linear model accurately describes the rate of taste bud regeneration, the first buds must have appeared 16 days after GLX (i.e. the x-intercept was 16.15 days). At the very least, taste buds begin to reappear after GLX between 14 and 28 days after surgery.
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According to a one-way analysis of variance, the groups differed reliably in taste bud number [F(5,36) = 89.7, P < 0.001]. A Tukey—HSD post hoc test indicated that all groups had fewer taste buds than the CON (all P-values < 0.017). The number of taste buds seen 70 days after GLX was 80.3% that of CON.
Experiment 2
The three control groups did not differ in the total number of palatal taste buds [F(2,9) = 3.31, P = 0.08].
The total number of palatal taste buds seen even 224 days after GSPX was
considerably less than that seen in controls. The five cases in the GSPX/224
day group had 40, 54, 78, 84 and 216 taste buds. The latter case did have a
remarkable number of taste buds relative to the control average (278.0);
however, several observations strongly suggest that this animal had an
unsuccessful nerve transection on one side. First, the number of taste buds in
this rat was more than 2.5 times greater than that seen in the rat with the
second highest number of taste buds returning and was over 7 standard
deviations from the mean of the other four rats in the GSPX/224 day group.
Second, the distribution of taste buds on the palate (though not the NID) was
strikingly unilateral, with one half of the palate devoid of taste buds except
for a few near the midline. Although this pattern of denervation has not been
described in earlier reports of unilateral GSPX
(Cleaton-Jones, 1976;
Miller and Spangler, 1982), it
is difficult to find an alternative explanation than unilateral innervation
given that intact animals always display a relatively even bilateral coverage
of the palatine field in our own observations and in those reported by others
(Cleaton-Jones, 1976;
Miller and Spangler, 1982).
Finally, as described in the Materials and methods, following GSPX, rats in
our procedure reliably fail to recover their body weight until weeks after
surgery, whereas the case with 216 taste buds recovered body weight at a rate
comparable to controls, achieving its presurgical body weight on the eighth
day after surgery. For comparison, this animal's four cohorts in the GSPX/224
day group reached their presurgical body weight 21-41 days after surgery, and
CON/224 day rats did so 3-7 days after surgery. Based on this profile of
results strongly suggesting that the transection was incomplete on one side,
this outlying case was not included in the formal statistical analysis.
We found that, in contrast to the facility of lingual taste bud
regeneration after transection of the GL (Experiment 1) or CT
(St. John et al.,
1995), palatal taste buds regenerated after GSP transection at a
very slow rate (Figures 3 and
4). At 56 days, when the other
major gustatory nerves had induced considerable taste bud regeneration in
their respective receptor fields, an average of just 17 total taste buds had
returned to the palatal fields innervated by the GSP (6.1% relative to all
controls). Furthermore, although there were considerably more taste buds at
112 days (59 taste buds representing 21.2% relative to controls), the rate of
returning taste buds seemed to asymptote at this value because by 224 days
there was only an average of 64 buds (23.0% of controls). Apparently, for
whatever reason, the surgical method outlined here permanently prevents the
regeneration of over three-quarters the typical number of palatal taste
buds.
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When the soft palate and NID were examined separately, taste buds returned at roughly a similar rate in both receptor fields (Figure 5). Because the soft palate has far more taste buds than the NID, the percentage of returning taste buds is higher in the latter. By 224 days, the soft palate had an average of 28 taste buds (14.6% of the number seen in all controls), whereas the NID had 36 taste buds (41.8% of controls). In both fields, regeneration reached asymptotic levels by 112 days. Of the eight rats that had apparently achieved asymptotic regeneration (i.e. those at 112 and 224 days), four had more taste buds in the soft palate than the NID, three had more in the NID than the soft palate, and one had an equal number in each field.
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Interestingly, the control groups did differ in the number of taste buds in the soft palate [F(2,9) = 5.91, P = 0.02] but not in the NID [F(2,9) = 0.13, P = 0.88]. This finding underscores the poor regeneration seen in the 224 day group and suggests that there is no drop in taste bud number over this age span and perhaps even a small increase, at least in some receptor fields.
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Discussion |
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It is clear from this study and the work of others that the lingual gustatory nerves regenerate rapidly in the rat, promoting the reformation of taste buds in the appropriate receptor field. There is evidence that this regeneration is also accompanied by functional recovery as assessed behaviorally, electrophysiologically, and by the immunohistochemical staining of taste stimulus induced expression of immediate early genes in the rostral nucleus of the solitary tract (Cheal et al., 1977
; Barry et
al., 1993; Zuniga et al.,
1994,
1997;
St. John et al.,
1995; Cain et al.,
1996; Ninomiya,
1998; Barry, 1999;
King et al., 2000;
Kopka et al., 2000;
Saito et al., 2000;
Kopka and Spector, 2001; Saito
et al.,
2001a,b).
Apparently, the GSP does not have the same proclivity to regenerate as does
the CT and GL, at least not with our surgical approach. In recent years, the functional consequences of gustatory nerve transection have led to hypotheses concerning the organization of gustatory system. Because of the ease of the lingual taste nerves to regenerate, and the potential for the extent of regeneration to influence functional competence, histological analysis should always accompany such reports, particularly when postsurgical testing regimens necessitate long survival times. The present report provides important empirical information to guide the design of experiments studying the functional competence of animals with transected and regenerated gustatory nerves.
Source of innervation of regenerated taste buds
It is true that the present report provides no explicit verification that
the taste buds reappearing after surgery were induced by the regeneration of
the transected nerve as opposed to sprouting from another nerve. We made no
attempt to verify functional connectivity by recording from the transected
nerve during taste stimulation of the relevant receptor field. Indeed, there
are some reports that the lingual nerve proper can support the maintenance and
regeneration of some morphologically normal fungiform taste buds
(Kinnman and Aldskogius, 1988;
Hard af Segerstad et al.,
1989). Nevertheless, there is ample evidence in the rodent taste
system demonstrating that sprouting from other gustatory nerves does not occur
naturally, although experimentally cross-anastomosed nerves can induce taste
bud formation in their non-native field (Oakley,
1967,
1970;
Nejad and Beidler, 1987;
Ninomiya, 1998;
Smith et al., 1999).
Moreover, taste buds that reappear have been shown to be dependent on the
regeneration of the transected nerve (Cheal
et al., 1977; Barry
et al., 1993; Cain
et al., 1996;
Montavon et al.,
1996). In support of this, when the CT or GL are prevented from
regenerating, there is no significant increase in the number of taste buds
appearing in the denervated field over time
(King et al., 2000;
Kopka et al., 2000;
Kopka and Spector, 2001).
Regeneration of the CT
Previously, only the time course of taste bud regeneration in the anterior
tongue after bilateral CT transection had been examined parametrically
(Cheal and Oakley, 1977;
St. John et al.,
1995), although a partial time course for the other nerves can in
some cases be inferred by combining results from various studies in the
literature. St. John, Markison and Spector
(St. John et al.,
1995) found that after transection of the CT in the middle ear,
taste buds began reappearing between 2 and 4 weeks after nerve transection,
with asymptotic levels of two-thirds the control number of taste buds seen by
6 weeks. Although, in some cases, others have found a somewhat greater number
of taste buds returning (relative to controls) after long survival times [e.g.
80% (Kopka et al.,
2000)], there nevertheless may be a limit imposed on regeneration
by the number of intact, healthy fungiform papillae that remain. St. John
et al. (St. John et al.,
1995) found that the number of anterior tongue taste buds
increased, but the number of normal fungiform papillae decreased with time
since surgery. This time-dependent decrease in papilla number is supported by
data collected at single time points across studies reported in the literature
(Ganchrow and Ganchrow,
1989a,b;
Robinson and Winkles, 1991;
Smith et al., 1999;
Kopka et al., 2000).
Other investigators have demonstrated that either the CT or the taste buds
themselves appear to maintain the normal structure of the fungiform papillae;
for example, in the absence of the innervating nerve many fungiform papillae
develop ectopic filiform spines (Ganchrow
and Ganchrow, 1989a; Hard af
Segerstad et al., 1989;
Oakley et al., 1990;
Robinson and Winkles, 1991;
Oakley et al., 1993;
St. John et al.,
1995), and even in species where the denervated papilla maintains
an atrophied taste bud (the hamster), there are considerable morphological
changes in the pore region (Parks and
Whitehead, 1998). The degeneration of fungiform papillae is even
more pronounced with concomitant removal of the lingual branch of the
trigeminal nerve (Hard af Segerstad et
al., 1989) or if the CT is transected in the early postnatal
period (Sollars and Bernstein,
2000), and this is associated with proportionately fewer
regenerated taste buds after these manipulations.
Regeneration of the GL
Despite the considerable differences in the morphology of papillae that the
GL and CT innervate and the surgical method used to transect these two nerves,
the time-course data for the regeneration of taste buds innervated by these
nerves is remarkably comparable in some ways
(St. John et al.,
1995). With both nerves there was no evidence of reinnervation at
14 days, but in both cases taste buds began to return by 28 days. The first
appearance of taste buds after GL transection in our study is similar to that
seen in the rabbit, 25 days, reported by Fugimoto and Murray
(Fugimoto and Murray, 1970) and
the rat by Iwayama and Nada (Iwayama and
Nada, 1969). One difference between the time course of
regeneration between the CT and GL is that in the former case the return of
taste buds on the anterior tongue reached an apparent asymptote by 42 days
postsurgery, whereas return of vallate taste buds is linear through 70 days.
On the other hand, the number of taste buds seen 70 days after GLX was 80.3%
that of our sham-operated controls, which is virtually the same extent of
regeneration seen in anterior tongue taste buds after CT transection after a
long postsurgical survival time (>84 days) in another study
(Kopka et al.,
2000).
As discussed above, there is correlational evidence that CT
transection-induced degenerative changes in fungiform papillae might
contribute to the asymptotic levels of taste bud reformation seen 42 days
after nerve transection. Like the fungiform papillae, the vallate papilla
undergoes morphological changes following denervation (Guth,
1957,
1963;
Kennedy, 1972;
State, 1977). Whereas Kennedy
found a thickening of the epithelium formerly containing taste buds
(Kennedy, 1972), Guth (Guth,
1957,
1963) and State
(State, 1977) found a
pronounced atrophy. The reason for this difference is unclear. Whether the
degenerative changes represent a progressive process awaits further anatomical
scrutiny. Thus, whereas there is a reasonable relationship between the time
course of fungiform papillae degeneration and the compromised ability of the
regenerated CT nerve to re-form taste buds (fueling speculation of a causal
relationship), it is premature at this time to posit a similar relationship
between degenerative changes in the vallate papilla and the extent of taste
bud reformation after GL transection.
In the current study, the rate of taste bud regeneration was strikingly
linear (Figure 2), but, at the
same time, only 80.3% of the control number of buds were seen 70 days
post-surgery. If the number of buds were to continue to return in linear
fashion, the vallate papilla would regain normal numbers of taste buds 87.3
days after surgery. Alternatively, it is possible that, like the fungiform
receptor field, the vallate papillae does not return to preexisting numbers of
taste buds. In a previously published study using identical surgical and
histological methods, in which the strain of rat, diet and housing conditions
were consistent with the present study, and in which taste pores were
quantified by a common experimenter (M.G.), the number of taste buds in the
vallate papilla was significantly less than in GL-intact rats 94 days after
transection [data from King et al.
(King et al., 2000),
replotted in our Figure 2].
Thus, had the present study been carried out to another data point, evidence
for an asymptote may have been obtained.
The rate and extent of regeneration in our rats appears to be quite
consistent with a parametric analysis of a group reported by Hosley et
al. (Hosley et al.,
1987a). In their AV75/CR75 group, 75-day-old rats (i.e. adults
similar in age to those of the present report) received avulsion of the right
GL in a manner that prevented regeneration, and a crushing of the left GL in a
manner that promoted regeneration. In their study, vallate taste buds
reappeared in a more or less linear fashion over the next 75 days (at
virtually the same rate as in our rats, six or seven buds per day), achieving
66.4% of the normal complement of their `normal' rats (610 buds, somewhat
higher than our CON group). Because a unilateral GL can maintain
greater than 80% of the rat vallate taste buds
(Guth, 1963) due to the
nerve's bilateral distribution in the papilla
(Whiteside, 1927;
Oakley, 1974;
Hosley et al.,
1987b), the time course data from the AV75/CR75 group in the
Hosley et al. (Hosley and Oakley,
1987; Hosley et al.,
1987a,b)
experiment would appear to be quite consistent with our own bilateral
transections.
Consistent with the conclusions of studies on the development of the
GL-vallate system (Hosley et al.,
1987a,b;
Hosley and Oakley, 1987;
Oakley, 1993), our data
suggest that the time course of regeneration in adult rats is somewhat
different from initial formation of taste buds during development. Although
the overall time course over which taste buds appear is quite similar to that
during development [i.e. 75-90 days
(Hosley and Oakley, 1987)],
during development the appearance of taste buds is not approximately linear,
but rather follows a third-order function characterized by an early
accelerated accumulation of buds (13 taste buds per day over postnatal
days 3-33), and a later deceleration in the accumulation of buds (just two
taste buds per day over postnatal days 60-90). To what extent this difference
reflects the different states of the developing and adult-transected papilla,
differences in the rate that axons reenter the papilla, or other factors
cannot be discerned from our data.
Regeneration of the GSP
In contrast to the CT and GL, transection of the GSP in the middle ear caused a pronounced and seemingly permanent decrease in the taste bud count in its receptor field. After 224 days, only 25% of the normal complement of palatal taste buds had regenerated, a number not significantly different from that seen at 112 days. Thus, this may represent the maximal amount of regeneration to be expected from our surgical approach. The case with the most taste buds at either 112 or 224 days had just 38.8% that seen in the average of all 12 controls.
What accounts for the poor percentage of regenerated taste buds after GSPX
relative to the nearly complete regeneration after transection of the other
major gustatory nerves? The target organ of the GSP (the palate) is different
from that of the other gustatory nerves (the tongue). It is possible that
denervation in adulthood renders these epithelia nonconducive to the formation
of taste buds as is the case with neonatal avulsion of the other gustatory
nerves (Hosley et al.,
1987a,b;
Sollars and Bernstein, 2000).
We did not make morphometric analyses of the palatal epithelium in the current
study, and, to our knowledge, epithelial changes after GSPX have not been
examined as they have been for the other gustatory nerves.
A more relevant factor, however, may be the nature of the surgical approach
necessary to transect the GSP. Whereas transection of the CT and GL can both
be relatively benign with regard to nearby structures, our exposure of the GSP
involved considerable disruption of the tissue surrounding the nerve (see
Materials and methods). Perhaps the approach used to transect the GSP produced
scars that impeded regenerating fibers. In support of this, Kopka et
al. (Kopka et al.,
2000) successfully prevented regeneration of the CT by cauterizing
structures of the external and middle ear to stimulate the secretion of
cerumin which fills the cavity of the bulla.
It should be noted that others have apparently been able to achieve
regeneration of palatal taste buds after a cross-anastomosis of the proximal
stump of the transected CT and the distal stump of the transected GSP
(Nejad and Beidler, 1987).
Likewise, these same investigators were able to form a successful
cross-regeneration of anterior tongue taste buds by bridging the proximal
stump of the transected GSP with the distal stump of the transected CT.
Although the presence of taste buds was not quantified, these investigators
were able to record electrophysiological responses in the cross-regenerated
nerves to taste stimuli applied to the appropriate receptor field. Thus, it
would appear that the GSP, under some circumstances, does have the capacity to
regenerate. Likewise the palate, under some circumstances, appears to be able
to support the reformation of new taste buds following a period of
denervation.
The GSP normally innervates three receptor fields: the posterior palatine
field and geschmacksstreifen (both on the soft palate), and the NID of the
hard palate (Cleaton-Jones,
1976; Miller,
1977). Soft and hard palate taste buds returned at the same rate,
and if anything, the NID were preferentially innervated by the returning GSP
fibers. Whether this means that certain axons within the GSP are `targeted' to
one specific receptor field (e.g. the NID) rather than accepting the first
available denervated epithelial tissue remains to be determined.
Concluding remarks
Histological analysis following gustatory nerve transection is critical for
the interpretation of behavioral studies, because rodent gustatory nerves are
noteworthy for their propensity to regenerate rapidly. This is especially
important in cases where behavioral tasks require long-term testing following
surgery [e.g. detection threshold testing
(Spector et al.,
1990)]. These data also provide a framework for experiments
designed to examine functional or anatomical consequences of completely
regenerated gustatory structures, or attempting to relate the extent of
functional recovery in incompletely regenerated structures. The current study
was an attempt to fill gaps in the literature on the time course of
regeneration of taste buds after bilateral transection of the major gustatory
nerves. While specific details will of course vary with surgical approach
[e.g. crushed gustatory nerves regenerate more rapidly than transected nerves
(Cheal and Oakley, 1977)], we
have nonetheless uncovered some useful parameters enabling the investigator to
temporally predict the extent of taste bud regeneration following gustatory
nerve transection.
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Acknowledgments |
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We would like to thank Dr Camille King for providing the 94-day data for Figure 2 and Lee Hallagan and Sara Saperstein for reading an earlier version of this manuscript. Supported in part by a grant from the National Institute on Deafness and Other Communication Disorders (R01-DC01628).
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Accepted October 28, 2002
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