Gustatory dysfunction in relation to circumvallate papilla’s taste buds structure upon unilateral maxillary molar extraction in Wistar rats: an in vivo study

Background: The interaction between taste sensation and dentoalveolar innervation is still under research. teeth loss can alter taste thresholds in humans, but the underlying mechanisms are still obscure. This study investigated the effect of unilateral maxillary molars extraction on the structure of circumvallate papilla in rats. Methods: Thirty-two male Wister rats, aged 3-4 months were randomly distributed into four groups (one control and 3 experimental ) each including 8 animals. The rats were euthanized 3, 6 or 9 weeks following the procedure. The changes in trough length and the taste buds structure and number of both sides of CVP were investigated using routine histological examination followed by statistical analysis. Results: the trough toward the extraction side was obviously shorter with a noticeable decrease of taste buds’ number than the non-extraction side. Taste buds were reduced in size and most of them showed signs of degeneration which was more evident in group II followed by group III, less deformity detected in group IV in comparison to the preceding 2 experimental groups. the non-extraction side of all experimental groups showed normal trough length and generally normal histology of taste buds. Conclusions: Maxillary molars extraction has a degenerative effect on the structure of taste buds and gustatory epithelium which were more marked at the extraction side and showed improvement upon elongation of follow up period


Introduction
Taste is an essential sensation that is mediated via taste buds (TBs) which are specialized neuroepithelial structures distributed predominantly over the dorsal surface of the tongue 1-3 , and undergo continuous renewal throughout adult life 4,5 .
The circumvallate papilla (CVP) is a major compartment of the gustatory system in the tongue that harbors more than 500 taste bud openings in its trough 6,7 . This gathering of taste organs in one specified place, make it easier to detect different changes under study.
Moreover, the impact of nerve injury on fungiform taste buds appears to be less severe than on circumvallate taste buds, thus making it a good candidate to study neural variations 8,9 .
The gustatory system is unique in that taste buds innervation is essential to taste bud cell turnover 10 . In the rat tongue, the single central CVP is innervated by the glossopharyngeal nerve bilaterally 7,11 . Taste sensation is modulated by different factors including the integration of gustatory information with other sensory modalities such as olfaction, food texture, temperature and pain (hot spices) through the trigeminal nerve 12 . Other factors include nerve supply and mechanical stimuli that are affected by the integrity of masticatory apparatus 13 .
Taste signals synapse to the nucleus of the solitary tract (NST) located in the medulla 14 . An interaction between dental and gustatory afferent neurons in the NST has been previously discussed 9,15-17 . In addition, teeth loss can alter taste thresholds in humans, but the underlying mechanisms are still obscure 15,18,19 .
This interaction has clinical implications as occlusion impairment by teeth extraction (dental deafferentation) can be considered a possible cause of taste alteration 15 .
It was reported that after unilateral transection of glossopharyngeal nerve in rat, the TBs loss in CVP was 12% rather than a 50% which indicate bilateral innervation of the remaining taste buds 20 . However, another study suggested that all the TBs in the rat CVP are bilaterally innervated except the upper two thirds of the outer trough walls which are innervated by ipsilateral glossopharyngeal nerve 21 .
The purpose of this study was to compare between both sides of the CVP regarding trough length and histopathological changes in the TBs structure and number secondary to unilateral maxillary molars extraction and to identify if unilateral extraction will cause damage to glossopharyngeal nerve on the same side of CVP only or may extend to the other side.
Rats were the animal of choice for this experiment because of their similarity to human anatomy regarding turnover time of taste buds, and innervation pattern of the tongue 12,13 .

Animals
A total of 32 male Wistar rats weighing 150-200 g, aged 3-4 months were used in this study. The animals were obtained from and housed in the Animal House, Faculty of Medicine, Cairo University. The animals were maintained in an air conditioned animal house under controlled room temperature 25±2°C with 12/12 h light/dark cycle and were allowed unlimited access to powdered soft food and water. Rats were housed in (standard polycarbonate cages (Pretty industries, Model:CR5). Control rats were placed as 4 animals per cage, while the experimental animals were placed one per cage until wounds had fully recovered in order to control infection.
All procedures were refined to reduce the negative impact on animals including administration of anesthesia prior to the procedure and gentle handling of the animal during extraction to prevent any tissue laceration or damage. After the experiment the animals received antibiotics to prevent infection, and analgesics to manage any pain or discomfort. The cages were cleaned frequently, they were supplied with wood chips substrate, colored tubes and ropes to ensure enriched environment for the animal.
The welfare of the animals was assessed prior to, during and after the experimental period by the attending veterinarian who intervened in case of pain or distress signs according to the ethical protocols for animal treatment that were supervised by the animal facilities, Faculty of Medicine, Cairo University. The study was approved by the Institutional Animal Care and Use Committee, Cairo University (Approval no. CUIIIS1016).

Sample size calculation
Sample size calculation based on previous research by Hsu et al., 2014; that reported the difference in the trough depth between the control and experimental (28 days) groups was 28 ±13 µm. Using power 90% and 5% significance level, 6 rats in each group was calculated to be sufficient to be able to reject the null hypothesis that the population means of the experimental and control groups are equal. That number was increased to 8 rats in each group to compensate for possible loss of animals during breeding. Sample size calculation were performed using PS: Power and Sample Size Calculation software Version 3.1.2 (Vanderbilt University, Nashville, Tennessee, USA).
Animal grouping and study design Rats were randomly distributed by Random Sequence Generator program (randomizer.org) into four groups (one control and 3 experimental) each including 8 animals. Implementation of the allocation was done as follow: numbers from 1 to 32 were written on folded papers that were placed in opaque sealed envelopes, matching of the rats with the numbers were done blindly through the technician in charge at the animal house, each rat was attached to its number till the end, then the numbers were opened and the rats were allocated in their groups according to the program recommendations.
The control (group I) were anesthetized without tooth extraction (sham operation) and euthanized after 3 weeks. In the experimental groups, all maxillary molars of the right side were extracted (early in the morning) under general anesthesia by an intraperitoneal injection of ketamine/xylazine (ketamine 40-100 mg/kg IP/ xylazine 5-13 mg/kg IP, Trittau, Germany) for 60-80 minutes 22 . Extraction were performed using Halsted Curved Mosquito Hemostatic Forceps, Germany 15921-G with the tongue retracted by a tweezer. Postoperatively, antibiotics were administrated (cefotaxime10mg/Kg IV, Eipico, Egypt) for 3 days 23 . Analgesics were administrated (Ketoprofen (Anafen, Merial) 5mg/kg I.M) 24 once daily for up to 3 days.
The first euthanization date for the current study (3 weeks) were assigned according to Huang & Lu, 1996 study as they found that taste buds of the CVP were diminished 3 weeks after neurectomy 26 . The other intervals (6 and 9 weeks) were set in order to investigate the probability of recovery from any potential damage. The CVP was dissected out according to the animal's tongue anatomy. The CVP is situated most posteriorly at the center of the base of the tongue, the site of the CVP in the dissected rat tongue was identified, then the tissue specimen was isolated by a horizontal cut anterior to the papilla and two oblique cuts posteriorly. Tissues were fixed in 10% calcium formol (No:8012-95-1, El-Gomhouria co., Egypt).
The sections were deparaffinized and rehydrated by immersing them successively for ~5 min with agitation in xylene, 100% ethanol, and 70% ethanol. Then the slides were rinsed in running tap water at room temperature for at least 2 min. The sections were stained in hematoxylin solution for 3 min then placed under running tap water for 1 min. The sections were stained in 1% eosin Y solution for 2 min. The sections were dehydrated with two consecutive immersions in 95% alcohol, then two immersions in 100% alcohol for 30 sec each. The alcohol was then extracted with two consecutive immersions in xylene 27 .
Blinding was applied for the primary animal care giver and the assessor of the histological and statistical results.
The specimens were examined using Leica DM300 light microscopic (Leica Microsystems, Inc., Switzerland).

Histomorphometric analysis
TBs' number for each specimen were counted using an objective lens of magnification (x40) for all groups by light microscope. TBs were marked by green crosses and counted by Image J Version:1.52p computer system ( Figure 1).
The length of both trough sides for each specimen was measured using an objective lens of magnification (x10) for all groups using light microscope. The length was measured using computer system ( Figure 2).
The trough opening was determined by a solid line drawn between the 2 troughs, the trough depth was marked by two lines, from top to bottom.

Statistical analysis
Comparisons between groups were performed using one-way analysis of variance (ANOVA). This was followed by Tukey's post hoc test for pairwise comparisons when ANOVA revealed a significant difference. Independent t test was used to compare both trough sides using IBM SPSS 18.0 version 21 for windows (SPSS Inc., Chicago, IL, USA). The significance level was set at p ≤ 0.05.

Histological results
Analysis of the control group samples revealed normal CVP surrounded by even trough lengths with normal TB histology and number (Figure 3). In groups II and III, the papillae showed slight deformity of the general outline as the trough toward the extraction side was obviously shorter with noticeable decrease of TBs' number than the non-extraction side. TBs were reduced in size and most of them showed signs of degeneration which was most evident in group II, followed by group III (Figure 4, Figure 5). A similar situation was observed in group IV with less deformity of the general outline, as the right side revealed an increase in length with apparent increase in TB number in comparison to the preceding two experimental groups. TBs exhibited nearly normal shape, with some degenerated taste cells ( Figure 6). TBs of the non-extraction side in all experimental groups showed generally normal histology except for slight enlargement and few darkly stained nuclei ( Figure 7). Raw microscope images are available as underlying data [28][29][30][31] .
Statistical results TB number and trough length variation between the two sides (extraction & non-extraction) were recorded among all experimental groups. It was noticed that non-extraction side recorded a higher mean number and length within each group, while the highest mean value was recorded in group II. This difference was not statistically significant in the controls, but was significant in groups II, III & IV (p ≤ 0.05). (Table 2 and Table 3, Figure 8 and Figure 9, underlying data 32 ).

Discussion
Taste is a highly dynamic and complex process. The interaction between gustation and somatosensation of the tongue is still under investigation 13 . The consequences of dental deafferentation as one of the possible causes of taste disorders, is unclear and therefore an area for further investigation. On the other hand, Guth 1963 found that following unilateral glossopharyngeal nerve transection in the CVP of rats, the mean loss of taste buds in the outer trench walls was greater on the operated than the unoperated sides. However, they found that this difference was not statistically significant 20 . Guagliardo & Hill, 2007 demonstrated that TBs of fungiform papilla on the uncut side increased in size 34 . This might explain enlarged TBs on the non-extraction side.
The highest mean values were recorded in group II towards the non-extraction side, which could be explained as a compensatory mechanism for the damage that has occurred at the other side.
Improved results observed in group IV regarding trough length and TBs' number are probably due to the recovery of the gustatory epithelium with time. This is in agreement with Lim & Green, 2008 who postulated that nerve damage in certain region would augment remaining taste signals in the neighboring area 35 .
Study limitations may include that the process of counting TBs, which needs to be more precise, as the same taste bud may appear in different sections, like counting the total number of taste buds per papilla by scoring only TBs of maximum dimensions. A more accurate investigation methods could be used in further studies rather than mere TBs counting, like using reverse transcription polymerase chain reaction (RT-PCR) for detection of the expression of certain genes.
The current study has clinical implications, as it identified a correlation between normal occlusion and taste sensation, and it could be considered as an aid in explaining the etiology of taste loss. According to the current study results, it is important to screen subjects for dental status when planning taste studies, especially in sensory analyses. Subjects with recent multiple extractions should not be included in such studies as taste sensation is likely to have been affected. These findings also could help to distinguish the effect of normal aging from disease-related changes in taste perception.
The current study provides a step forward in predicting the effect of molar extraction and the impact of unilateral glossopharyngeal damage on taste sensation in humans. However, further investigations and observational studies are needed to prove that connection.

Conclusions
Maxillary molars extraction has a degenerative effect on the structure of taste buds and the gustatory epithelium which were more marked at the extraction side, and showed improvement upon elongation of follow up period. : Introduction This section is well written but needs to be structured and reformatted into paragraphs rather than isolated sentences.

Data availability
:

Methods
First paragraph, there is an extra bracket () before standard polycarbonate cages that needs to be deleted. :

Results
Describing deformity of taste buds is very subjective and a more objective way of defining the degree of deformity is needed. Figure 4A is of a very poor quality and there are some concerns that it does not belong to this experiment at all due to different light conditions and degree/type of stain. It seems like a texbook image rather than an experimental one. Please replace this image.
Unfortunately, nothing was reported about the differences between the four groups using a one-way ANOVA. Even if there were no significant findings, it should still be reported.
ANOVA. Even if there were no significant findings, it should still be reported.
For the independent t-test results, please report the actual P value and standard deviations rather than (P<0.05). This is important to inform the readers about the degree of variability within your samples and the level of statistical significance. :

Discussion
It is recommended to discuss the difference that extraction of lower molars and wisdom teeth (in humans) would make to taste sensation as well. :

Conclusions
It is misleading to report that extraction of maxillary molars had a degenerative effect on circumvallate papillae in general as no data from this study supports this statement. The data from this study only shows degeneration in comparison to the non-extraction side. Also the claim that the degeneration improves by time is only an observation and is not supported by statistical analysis (No data from the one way ANOVA) was presented.
The conclusions need to rewritten both in the abstract and the full article text as it is over presenting the limited findings obtained from this study.

Are sufficient details of methods and analysis provided to allow replication by others? Yes
If applicable, is the statistical analysis and its interpretation appropriate? Partly Minor comments would help to improve the impact of this paper:

Title:
Gustatory dysfunction in relation to circumvallate papilla's taste buds structure upon unilateral maxillary extraction in Wistar rats but in you said (all maxillary molars of the right side were molar Methods extracted) so you may change it to maxillary molars.

Introduction:
An interaction between dental and gustatory afferent neurons in the NST has been previously please add a hint about it discussed

Methods:
In the experimental groups, all maxillary molars of the right side were extracted (early in the morning) why? Is the time of extraction make a significant difference.
The tongue retracted by a tweezer. Postoperatively, antibiotics were administrated (cefotaxime10mg/Kg IV, Eipico, Egypt) for 3 days23. Analgesics were administrated (Ketoprofen (Anafen®, Merial) 5mg/kg I.M)24 once daily for up to 3 days did you do that with the control group? If not how to be sure that the medications does not affect the experiment.

Results:
Figures from 2 to 6: please clarify which side is the extracted molars side and adjust the angulation of the CVP (figure 3 and 5) to make it easy to compare between the 2 sides of the trough.

Discussion
Further investigations of the nerve supply under TBs of both sides using immunohistochemistry is recommended.

Are sufficient details of methods and analysis provided to allow replication by others? Yes
If applicable, is the statistical analysis and its interpretation appropriate? I cannot comment. A qualified statistician is required.
Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes No competing interests were disclosed. Competing Interests: Reviewer Expertise: Oral biology, oral histology, immunohistochemistry, oral anatomy and dentistry I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
The benefits of publishing with F1000Research: Your article is published within days, with no editorial bias You can publish traditional articles, null/negative results, case reports, data notes and more The peer review process is transparent and collaborative Your article is indexed in PubMed after passing peer review Dedicated customer support at every stage For pre-submission enquiries, contact research@f1000.com