Evaluating Instantaneous Perfusion Responses of Parotid Glands to Gustatory Stimulation Using High-Temporal-Resolution Echo-Planar Diffusion-Weighted Imaging

BACKGROUND AND PURPOSE: Parotid glands secrete and empty saliva into the oral cavity rapidly after gustatory stimulation. However, the role of the temporal resolution of DWI in investigating parotid gland function remains uncertain. Our aim was to design a high-temporal-resolution echo-planar DWI pulse sequence and to evaluate the instantaneous MR perfusion responses of the parotid glands to gustatory stimulation. MATERIALS AND METHODS: This prospective study enrolled 21 healthy volunteers (M/F = 2:1; mean age, 45.2 ± 12.9 years). All participants underwent echo-planar DWI (total scan time, 304 seconds; temporal resolution, 4 s/scan) on a 1.5T MR imaging scanner. T2WI (b = 0 s/mm2) and DWI (b = 200 s/mm2) were qualitatively assessed. Signal intensity of the parotid glands on T2WI, DWI, and ADC was quantitatively analyzed. One-way ANOVA with post hoc group comparisons with Bonferroni correction was used for statistical analysis. P < .05 was statistically significant. RESULTS: Almost perfect interobserver agreement was achieved (κ ≥ 0.656). The parotid glands had magnetic susceptibility artifacts in 14.3% (3 of 21) of volunteers during swallowing on DWI but were free from perceptible artifacts at the baseline and at the end of scans on all images. Increased ADC and reduced signal intensity of the parotid glands on T2WI and DWI occurred immediately after oral administration of lemon juice. Maximal signal change of ADC (24.8% ± 10.8%) was significantly higher than that of T2WI (−10.1% ± 5.2%, P < .001). The recovery ratio of ADC (100.71% ± 42.34%) was also significantly higher than that of T2WI (22.36% ± 15.54%, P < .001). CONCLUSIONS: Instantaneous parotid perfusion responses to gustatory stimulation can be quantified by ADC by using high-temporal-resolution echo-planar DWI.

Q uantification of normal salivary gland function is of paramount clinical importance because it is the foundation of accurately evaluating disease-related salivary gland functional impairment. Salivary gland function can be estimated by several methods such as saliva collection, 1 laboratory measurement of the chemical and biochemical components, 2 scintigraphy, 3 singlephoton emission CT, 4 and positron-emission tomography. 5 MR imaging is superior to saliva collection and laboratory and biologic studies by providing morphologic and functional information of the parotid glands simultaneously and specifically for individual salivary glands. On the other hand, MR imaging is also superior to scintigraphy, SPECT, and PET because of its high spatial resolution and radiation-free nature.
In recent decades, DWI has been increasingly applied to probe salivary gland function in addition to evaluating tumors, 6-11 connective tissue disorders, 12 Sjögren syndrome, 13,14 and postradiotherapy change [15][16][17][18][19] of the parotid glands. However, 2 mutually opposed trends of parotid apparent diffusion coefficient changes after gustatory stimulation have been observed in different study groups, even in healthy volunteers. While some researchers demonstrated an increase of parotid ADC after gustatory stimulation, 14,16,20-23 others showed a decrease of parotid ADC after this stimulation. 15,24 Such discrepancy has been partially attributed to the different types and dosages of the stimulators. 16 Nevertheless, the discrepancy of diffusional responses to gustatory stimulation in the aforementioned DWI studies has raised concern for whether the normal salivary gland function has been evaluated appropriately with DWI.
The role of the temporal resolution of DWI, which might potentially influence researchers in interpreting parotid gland function, has not been documented to date, to our knowledge. Via parasympathetic innervation, salivary glands secrete and empty saliva into the oral cavity rapidly after gustatory stimulation. Current DWI studies might have limitations in catching the instantaneous responses of the parotid glands due to insufficient temporal resolution. We hypothesized that the parotid glands respond to the oral administration of lemon juice on the order of seconds. The aim of our study was to design a high-temporal-resolution echo-planar pulse sequence for DWI and to quantify the instantaneous MR perfusion responses of the parotid glands to gustatory stimulation.

MATERIALS AND METHODS
This prospective study was approved by the institutional review board at Tri-Service General Hospital. Written informed consent was obtained.

Subjects
This study initially enrolled 22 healthy volunteers who were free from tumor, inflammation, autoimmune disease, operations, and radiation therapy involving any parotid gland. One subject was excluded due to severe imaging distortion related to a fixed metallic dental brace. Finally, 21 healthy volunteers were enrolled, including 14 men and 7 women (mean age, 45.2 Ϯ 12.9 years). Saliva production of each volunteer was quantified 1 hour before DWI by using the Saxon test. 1

MR Imaging Protocol
All MR images were performed on a 1.5T whole-body scanner (Signa HDx; GE Healthcare, Milwaukee, Wisconsin) by using an 8NV head and neck array coil. Three-plane orthogonal gradientecho images were acquired for anatomic localization. Single-shot echo-planar DWI (TR/TE/NEX, 2000/53.3 ms/1) with fat saturation was performed on axial planes with diffusion-probing gradients (bϭ0 and 200 s/mm 2 ) applied on each of 3 orthogonal directions alternatively. We intentionally chose a b-value of 200 s/mm 2 for 3 reasons: First, a b-value higher than 200 s/mm 2 has been shown to be perfusion-insensitive in ADC measurements. 25,26 Accordingly, choosing a b-value of 200 s/mm 2 allows better evaluation of perfusion-sensitive changes of the parotid glands than by using a b-value of 300 s/mm 2 or higher. Second, apparent bulk motion artifacts have been demonstrated in dynamic scans on DWI with a high b-value of 1000 s/mm 2 in the parotid glands. 27 On the contrary, DWI with a low b-value of 200 s/mm 2 has a higher signal-to-noise ratio 28 and is theoretically less susceptible to bulk motion artifacts than with higher b-values. Third, although it has been documented that b-values lower than 200 s/mm 2 are critical to obtain perfusion-sensitive information from DWI, there is no consensus on the magnitude of b-values that should be applied. 29 We chose a b-value of 200 s/mm 2 rather than 100 s/mm 2 to reduce potential contamination of signal loss from the faster flow of small arteries or veins. Other MR imaging parameters included an FOV of 240 ϫ 240 mm, matrix size of 256 ϫ 256, echo-train length of 76, bandwidth of 1953 Hz/pixel, and section thickness of 5 mm. Each DWI examination contained 152 excitations. Each excitation obtained 14 sections covering from the cerebellum to the submandibular glands. The total scan time was 5 minutes 4 seconds.

MR Imaging Acquisition
T2WI (b ϭ 0 s/mm 2 ) and DWI (b ϭ 200 s/mm 2 ) were acquired alternatively. The first 2 excitations were discarded. The direction of the diffusion gradients was changed periodically in the order of the x, y, and z axes, allowing acquisition of a series of T2WI-DWI x -T2WI-DWI y -T2WI-DWI z scans for the rest of the 150 excitations as illustrated in Fig 1A. Accordingly, 75 dynamic scans were used for analysis with each dynamic scan containing a T2WI and a DWI. The temporal resolution was 4 seconds per scan.

Gustatory Stimulation by Lemon Juice
As shown in Fig 1B, 10 mL of commercial lemon juice was administrated via a connecting tube into the oral cavity at the 11th dynamic scan. Each subject was instructed to swallow the lemon juice at the 21st scan. The duration of lemon juice stasis in the oral cavity was 40 seconds.

Qualitative Assessment of Imaging Quality
All MR imaging data were processed by software developed inhouse (T.-W.C., Y.-H.L., and C.-J.J.) by using Matlab (Math-Works, Natick, Massachusetts). Magnetic susceptibility artifacts on T2WI and DWI were qualitatively and independently evaluated by 2 neuroradiologists (C.-J.J. and C.-W.W. with Ͼ10 and 3 years' experience in head and neck MR imaging interpretation, respectively) by using a 4-point grading score system (0, severe magnetic susceptibility artifacts: distortion and signal loss that The first 2 images were discarded. T2-weighted images (b 0 ) and diffusion-weighted images (b 200 ) were arranged alternatively for 75 dynamic scans. For DWI, diffusion gradients were applied along the x, y, and z axes periodically. B, Lemon juice was introduced into the oral cavity via a connecting tube at the 11th scan and swallowed at 21st scan. Figure 1B is courtesy of Cheng-Hsuan Juan.
involved the entire image; 1, moderate magnetic susceptibility artifacts: distortion and signal loss involving the parotid glands; 2, mild magnetic susceptibility artifacts: distortion and signal loss involving the oral cavity, nasal cavity, oropharynx, maxillary sinuses, or masticator spaces but sparing the parotid glands; and 3, no magnetic susceptibility artifacts). Qualitative analysis was performed on T2WI and DWI at baseline, during swallowing, and at the end of dynamic scans, respectively.

Imaging Processing and Quantitative Data Analysis
Quantitative analysis was performed on 3 contiguous sections covering the largest areas of the parotid glands, respectively. Polygonal ROIs were drawn within the bilateral parotid glands on the T2WIs, respectively, avoiding the partial volume effects from visible vessels and adjacent tissues. These ROIs were then automatically copied to the DWI for concurrent measurement of signal intensity. Time signal data of T2WI and DWI were treated by a fifth-order Butterworth low-pass filter with a cutoff frequency of 0.025Hz (Fig 2) to eliminate high-frequency noise that occurred during the dynamic scans.
ADC was calculated on the basis of the following equation: where S 200 and S 0 were signal intensities of images with b-values of 200 s/mm 2 and 0 s/mm 2 , respectively. Signal intensity-time curves for T2WI, DWI, and ADC maps were plotted. In addition, signal intensity-time curves were further normalized according to equation 2: where SC n was the normalized signal change, SI i was the signal intensity at the ith scan, and SI 0 was the baseline signal intensity averaged from the first 9 (first to ninth) scans. Salivary parameters, including maximal signal change (MSC), time to peak, and recovery ratio (RR), were further derived from the normalized signal change-time curves, respectively, to characterize the indi-vidual parotid responses to gustatory stimulation. MSC was defined as the maximal normalized signal change, TTP referred to the time interval from the start of dynamic scans to the time of MSC, and RR was calculated according to equation 3: where SC end refers to the normalized signal change averaged from the last 9 (67th to 75th) scans.

Statistical Analysis
Statistical analysis was performed by using SPSS software (SPSS 20.0; IBM, Armonk, New York) and MedCalc for Windows (MedCalc Software, Mariakerke, Belgium). Interobserver reliability for imaging distortion was evaluated by linear-weighted statistics. The normality of baseline signal intensity and salivary parameters was analyzed by Kolmogorov-Smirnov tests. A paired Student t test was used for comparisons between the left and right parotid glands. Salivary parameters were analyzed by 1-way ANOVA; and post hoc group comparisons, with a Bonferroni correction. A P value Ͻ .05 was statistically significant.

RESULTS
The saliva collected within 2 minutes was 5.78 Ϯ 2.61 g (mean Ϯ standard deviation). Results of qualitative analysis of T2WI and DWI were summarized in the Table. Linear-weighted analysis revealed substantial agreement between the 2 raters with a value of 0.656 on all T2WIs and almost perfect agreement, with a value ranging from 0.842 (during swallowing) to 1 (at baseline and at the end of dynamic scans) on DWI. The parotid glands were free from perceptible artifacts at the baseline or at the end of scans on both T2WI and DWI (Fig 3). However, the parotid glands had magnetic susceptibility artifacts on DWI during swallowing in 14.3% (3 of 21) of volunteers who had metallic dental implants (Fig 4). The left parotid glands did not differ from the right parotid glands in baseline signal intensity on either T2WI (P ϭ .148) or DWI (P ϭ .227). Therefore, data of both parotid glands of each subject were averaged to represent each individual in further analysis. Averaged signal intensity-time curves of the parotid glands on T2WI, DWI, and ADC were plotted in Fig 5. On both T2WI and DWI, the signal intensity decreased immediately after oral administration of lemon juice and kept declining during the gustatory stimulation. After swallowing, the signal intensity began to increase slowly on DWI but further dropped without perceptible recovery on T2WI (Fig 5A). On the contrary, the ADC increased abruptly during the lemon juice stimulation, reached the peak at swallowing, and then declined to the baseline level at the end of scans (Fig 5B). Figure 6 demonstrates the normalized signal change-time curves of the parotid glands. The normalized signal change-time curves of the parotid glands on T2WI (Fig 6A), DWI (Fig 6B), and ADC ( Fig 6C) revealed trends similar to signal intensity-time curves, respectively. One-way ANOVA showed significant differences in all salivary parameters, including MSC, TTP, and RR among T2WI, DWI, and ADC, respectively (all, P Յ .001). The results of post hoc analysis with Bonferroni correction of salivary parameters are described below. The MSC of ADC (24.8% Ϯ 10.8%) was significantly higher than that of T2WI (Ϫ10.1% Ϯ 5.2%; P Ͻ .001) and DWI (Ϫ14.2% Ϯ 5.5%; P Ͻ .001). The MSC of T2WI did not differ from that of DWI (P ϭ .437). TTP was significantly higher in T2WI (47.0 Ϯ 17.3 scans) than in DWI (31.0 Ϯ 16.4 scans; P Ͻ .001) and ADC (21.6 Ϯ 11.1 scans; P ϭ .004). There was no difference in TTP between DWI and ADC (P ϭ .132). The RR of ADC (100.71% Ϯ 42.34%) was significantly higher than in T2WI (22.36% Ϯ 15.54%; P Ͻ .001) and DWI (42.3% Ϯ21.7%; P Ͻ .001). There was no difference in RR between the T2WI and DWI (P ϭ .143).

DISCUSSION
Temporal resolution is an important factor in analyzing immediate responses of the parotid glands to gustatory stimulation. Such immediate responses have not been emphasized in prior research, [14][15][16][20][21][22][23][24] in which DWI was performed without concurrent gustatory stimulation and the temporal resolution of DWI was rather low, ranging from 74 seconds per scan 21 to 162 seconds per scan. 16 Discrepant observations of parotid responses to the gustatory stimulation in prior research (ie, decreased ADC after gustatory stimulation in some studies 15,24 but increased ADC in others 14,16,[20][21][22][23] ) might be partly affected by the time gap between the gustatory stimulation and the poststimulation DWI and the low temporal resolution of DWI. Both factors limit DWI in detecting the maximal change of parotid ADC after gustatory stimulation.
In this prospective study, we successfully demonstrated the instantaneous responses of the parotid glands to gustatory stimulation by using echo-planar DWI at a high temporal resolution of 4 seconds. The overall signal intensity of the parotid glands on T2WI and DWI was altered at the start of stimulation, as was the parotid ADC. Our results support the hypothesis that the response of the parotid glands to gustatory stimulation occurs in seconds after oral intake of lemon juice.
On T2WI, the rapid reduction of signal intensity of the parotid glands might indirectly reflect an immediate reduction of saliva in the parotid glands after gustatory stimulation. Our result is consistent with the observations of quantitative salivary gland scintigraphic studies with a temporal resolution of 15 seconds per scan. 30 The immediate reduction of parotid radioactivity and accumulation of oral radioactivity following stimulation 30,31 supports saliva being emptied from the parotid glands immediately after the stimulation. After swallowing, the parotid glands showed persistent low signal intensity without recovery in averaged signal intensity-time curves and showed an RR of 22.36%, derived from individual signal intensity-time curves. Our results suggest that the refill of the water component of the parotid gland is a process longer than 3 minutes after removing the stimulator.
Because the signal intensity of vascular flow is attenuated rapidly under low b-values, 32 ADC calculated from low b-values (0 s/mm 2 and 200 s/mm 2 ) can be considered perfusion-sensitive in our study. Our study showed a maximal increase of 24.8% in the parotid ADC at 44.6 seconds (TTP ϭ 21.6 scans) after the start of lemon juice stimulation. Such a rapid increase of parotid ADC implies an instantaneous increase of parotid perfusion after gustatory stimulation. The parotid response to gustatory stimulation has been recently investigated by arterial spin-labeling perfusion-weighted MR imaging, 33 showing a mean increase of 62% of parotid blood flow. However, arterial  spin-labeling MR imaging is also limited in depicting the instantaneous response of the parotid glands due to its low temporal resolution of 160 seconds per scan. 33 Evaluation of parotid gland function by using blood oxygen level-dependent MR imaging showed an initial drop of signal intensity after gustatory stimulation by using ascorbic acid. 34 In addition to the aforementioned MR imaging techniques, dynamic contrast-enhanced MR imaging has also been applied to evaluate the perfusion change caused by salivary stimulation recently. 35 After swallowing, the parotid glands showed rapid reduction of ADC toward the baseline with an RR of 100% in our study. Our results reflect rapid reduction of parotid perfusion after swallowing lemon juice. The parotid ADC showed significantly higher RR than T2WI (P Ͻ .001). Our results suggest that the parotid perfusion has returned to the baseline level at the end of the dynamic scans, while the water refill of the parotid glands has not.
Our results also show apparent intersubject variations regarding all salivary parameters, especially after swallowing lemon juice. The intersubject variations might reflect the biologic diversity. If one takes TTP for example, possible reasons for the variations of TTP include residual lemon juice in the oral cavity, re-peated swallowing, and other involuntary motions or background noises during dynamic scanning.
Our study has several potential limitations. First, there is a trade-off between temporal resolution and signal-to-noise ratio in our study. Therefore, we performed an analysis of imaging quality to examine the severity of magnetic susceptibility artifacts. Our results showed that the parotid glands were free from magnetic susceptibility artifacts at baseline and at the end of dynamic scans with substantial-to-almost perfect interobserver agreement. Second, the signal intensity of DWI might be influenced by bulk motion in our study. Therefore, we applied a low-pass filter to reduce the high-frequency signal fluctuations. Our results are consistent with the aforementioned quantitative salivary gland scintigraphic studies 30,31 regarding rapid emptying of saliva and arterial spin-labeling MR imaging studies 33 regarding increased blood flow after gustatory stimulation. Third, signal intensity on T2WI and DWI might be not only attributed to water molecules but also affected by fat molecules. 36, 37 To verify the change of the water component in response to gustatory stimulation, another high-temporal-resolution echo-planar dual-echo MR imaging pulse sequence has been designed for direct measurement of the proton density of the parotid glands.

CONCLUSIONS
Instantaneous parotid perfusion responses to gustatory stimulation can be quantified by ADC by using high-temporal-resolution echo-planar DWI.