Fifty-five Sprague-Dawley (SD) rats (Samtako Co., O San, Korea) (mean age: 7 to 8 weeks, mean weight: 220 ± 30 g) were maintained at three rats per cage and they were given free access to standard rat chow and water. The rats were kept at a room temperature of 23 ± 1℃ and humidity of 55 ± 5%, with 12 hour lighting at 150-300 lux.

Our experiment is composed of two subexperiments. In the first experiment, 35 rats were used, and these were divided into seven groups according to a single radiation dose: the control (no radiation), 10 Gy, 15 Gy, 20 Gy, 25 Gy, 30 Gy, and 35 Gy groups of 5 rats per group. The rats were sacrificed 2 weeks later after X-irradiation by a barbiturate overdose. In the second experiment, 20 rats were exposed to 20 Gy, and they were divided into four groups of 5 rats each according to the elapsed time after radiation exposure: 3 days, 2 weeks, 4 weeks, and 12 weeks. Five rats of each group were also sacrificed by a barbiturate overdose to examine the effects of radiation on the body weight loss and the laryngeal muscles' MyHC isoform expression.

The head and neck areas of the rats were evenly irradiated with 2 Gy per minute by using a 6 MV energy therapeutic linear accelerator (CLINAC 600C, Varian, Palo Alto, CA, USA). To irradiate the head and neck area with a sufficient dose of radiation, a 1.0 cm bolus was used for increasing the radiation dose. A single dose of irradiation was administered. To immobilize the animals during their irradiation, the rats were anesthetized with an intraperitoneal injection of ketamin hydrochloride (40 mg/kg) and xylazine hydrochloride (5 mg/kg). No side effects or complications occurred during the rats' anesthesia.

Thirty-five rats were grouped into seven groups: the control group and the 10, 15, 20, 25, 30, and 35 Gy groups. Each group was composed of 5 rats. To immobilize the animals during their irradiation, the rats were anesthetized with an intraperitoneal injection of ketamin hydrochloride (40 mg/kg) and xylazine hydrochloride (5 mg/kg). All the animals were sacrificed two weeks later with a barbiturate overdose.

Twenty rats were equally grouped into four groups, and each group was irradiated with 20 Gy of X-ray. The rats in each group were anesthetized and sacrificed 3 days, 2 weeks, 4 weeks, and 12 weeks later, respectively. In general, we examined MyHC types I, IIa, IIb, IIx, and IIL in the TA, PCA, and CT muscles. We did not examine the effect of radiation on the TA expression of MyHC I because it has been reported that the TA muscle expresses very low levels of MyHC I.9 We also did not examine the expression of MyHC IIL in the CT muscle since its baseline expression of this isoform is very low.10

After resection of the larynx from the sacrificed rats by using a surgical microscope, the TA, PCA, and CT muscles were removed and stored in a - 80℃ freezer. The RNA was extracted on the same day.

The total RNA was extracted by using TRIzol® reagent (Invitrogen, Carlsbad, CA, USA) according to the method suggested by the manufacturer. The cDNA was synthesized from the RNA, by using Superscript III reverse transcriptase (Invitrogen), and RT-PCR was performed in a total reaction volume of 20 µL. In the first step, a mixture consisting of 1 µL random hexomer, 1 µL oligo (dT) 18, 1 µL 10 mM dNTP mix, 8 µL extracted total RNA and 2 µL DNase-RNase free water was reacted for 5 minutes at 65℃ and then it was cooled on ice for at least 1 minute. Thereafter, 4 µL of 5X first strand buffer, 1 µL of 0.1 M DDT, 1 µL Superscript III RT, and 1 µL RNase OUT (Invitrogen, Madison, WI, USA) were added. The mixture reacted at 25℃ for 10 minutes, 42℃ for 50 minutes, and 85℃ for 5 minutes. The residual complementary RNA was removed by adding 1 µL containing 2 units of E. coli RNAse H (New England BioLabs, Ipswich, MA, USA). After a reaction at 37℃ for 20 minutes, the samples were stored at -20℃ until use. The oligonucleotide primers for the MyHC type I, IIa, IIb, IIx, and IIL isoforms were synthesized (Table 1) by applying the Primer Express Program (Iowa city, IA, USA)which was published by the NCBI, to the MyHC gene sequences of the SD rats. Primers for the housekeeping control gene, i.e., glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were also used. All the primers we used were purchased from Bioneer Co. (Daejeon, Korea). Quantitative PCR was performed by using LightCycler® Software Version 4.0 and the LightCycler® Instrument (Roche, Seoul, Korea). A 20 µL total volume was prepared with 2 µL LightCycler® FastStart DNA Master SYBR Green I, 2.4 µL 25 mM MgCl2, 1 µL of each forward and reverse primer, and 2 µL cDNA and 11.6 µL DNase-RNase free water. For amplification, the reaction mixtures were placed on 32-well optical reaction plates and 50 cycles were performed. After the last cycle, melting curve analysis was performed to examine whether the products were the correct products. The LightCycler® (LightCycler® FastStart DNA Master SYBR Green I) PCR parameters are as follows: preincubation at 95℃ for 10 min, amplification, denaturation at 95℃ for 10 sec, annealing at 58℃ for 5 sec, and extension at 72℃ for 11 sec. The PCR parameters for the melting curve analysis are as follows: denaturation at 95℃ for 0 sec, annealing at 65℃ for 15 sec, melting at 95℃ for 0 sec, and cooling at 40℃ for 30 sec.

The comparative threshold method was applied to examine the effect of radiation on the MyHC expression in the laryngeal muscles of the SD rats. This method assesses the relative expression of the gene of interest by determining the difference between the threshold cycles of the gene of interest and the housekeeping gene. GAPDH served as the housekeeping gene. The 2^-ddCt method is well-known as a comparative threshold method and thus the following equation was used: ddCt = control group (Ct target-Ct reference) - irradiated group (Ct target-Ct reference). Two statistical analyses were performed for assessing the MyHC isoform expression. Paired t-tests with Bonferroni's correction was used to analyze the effect of different radiation doses and over time in the each MyHC isoform, and repeated measures of ANOVA (post-hoc analysis by Turkey) were used to analyze the differences between the MyHC isoforms in each muscle. p values less than 0.05 were considered statistically significant.

The rats exposed to 10-20 Gy lost weight soon after irradiation and they gradually recovered the weight lost. The rats exposed to 25 Gy or more radiation continuously lost weight before dying. In particular, the effect of 20 Gy radiation on the living rats was the most severe. These rats could not see properly and they had trouble opening their mouth to eat, so all the rats exhibited weight loss during the first 10 days of the experiment. However, they began to recover their weight after 2 weeks. The rats irradiated more than 20 Gy died between days 7 and 12 and they could not participate in the remainder of the study.

The TA, PCA, and CT muscles of the rats were dissected out separately and subjected to real time RT-PCR for assessing the expressions of the MyHC I, IIa, IIb, IIx, and IIL isoforms. Of the rats irradiated with 10, 15, or 20 Gy, most of the MyHC isoform expressions clearly showed a dose-dependent decrease, excluding the MyHC IIa expression in the TA and PCA muscles (Fig. 1). The statistical analysis using repeated measures of ANOVA (post-hoc analysis by Turkey) revealed that the MyHC IIa expression in particular significantly decreased, as compared to the expressions of the other MyHC isoforms, in all three muscles by 10 to 20 Gy radiation (p < 0.05) (Table 2).

The surviving 20 Gy-irradiated rats were examined 3 days or 2, 4, or 12 weeks after irradiation. The expression of almost all the MyHC isoforms by the TA, PCA, and CT muscles gradually decreased over time, except the MyHC I expression in the PCA and CT muscles and the MyHC IIL expression in the PCA muscle (Fig. 2). The TA, PCA, and CT muscle profiles were similar to the MyHC IIa expression as it rapidly declined from day 3 onwards and continued to decrease for up to 12 weeks. The statistical analysis using repeated measures of ANOVA (post-hoc analysis by Turkey) revealed that the MyHC IIa expression significantly decreased, as compared to the expressions of the other MyHC isoforms (p < 0.05) (Table 3).