(e) TUNEL-positive cells almost disappeared in the glands at 12 weeks after BTXA injection. acini. These results indicate that BTXA decreases the fluid secretion of submandibular glands and increases the concentration of amylase in saliva. Decreased manifestation of M3 receptor and AQP5, inhibition of AQP5 translocation, and cell apoptosis might involve in BTXA-reduced fluid secretion of submandibular glands. cell-death detection kit (Roche Applied Technology, Penzberg, Germany) was used according to the manufacturer’s instructions. Briefly, tissue sections were incubated with terminal deoxynucleotidyl transferase inside a humidified chamber at 37?C for 1?h. A mixture of antidigoxigenin-peroxidase and substrate-chromagen were utilized for visualization, and the cells were counterstained with hematoxylin. The nuclei of the apoptotic cells were stained dark brown. Reverse transcriptase polymerase chain reaction Total RNA from SMG was purified using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. cDNA was prepared from 5 g of total RNA with Moloney murine leukemia computer virus reverse-transcriptase (Promega, Madison, WI, USA). The sense and antisense primers for M3-muscarinic acetylcholine receptor (M3 receptor) (“type”:”entrez-nucleotide”,”attrs”:”text”:”NM_000740″,”term_id”:”1889688494″NM_000740), AQP5 (AF_495879) and -actin were 5-CTT CTC CAA GCT TCC CAT CCA-3 and 5-TGA CCG Take action GTC TCT GCT GGT-3, 5-GTT CCT GGC CAC CCT CAT CT-3 and 5-ACA GAC AGG CCG ATG GAC AG-3, and 5-ATC TGG CAC CAC ACC TTC TAC AAT GAG CTG GCG-3 and 5-CGC CAT Take action CCT GCT TGC TGA TCC ACA TCT GC-3, respectively.15 -actin was amplified as the internal standard. The amplification products were visualized on 1.5% agarose gel with ethidium bromide and sequenced to confirm their identities. The band densities were quantified using a LEICA-550IW image analysis system (Leica, Manheim, Germany). Immunofluorescence Frozen SMG sections were immunostained with anti-AQP5 antibody at a 1100 dilution, and incubated with FITC- or tetramethylrhodamine isothiocyanate-labeled secondary antibodies as explained previously.14 Nuclei were stained with 4,6 -diamidino-2-phenylindole (Sigma-Aldrich, St Louis, MO, USA). Fluorescence images were captured under a confocal microscope (TCS SP5; Leica, Heidelberg, Germany). Statistical analysis Data are offered as (means.d.). Variations among groups were analyzed by one-way analysis of variance, and then Bonferroni testing. A value of control. BTXA, botulinum toxin type A; SMG, submandibular gland. There were no significant variations between the weights of SMGs injected with BTXA and the normal control glands. The weights of SMGs was (0.490.08)?g, (0.550.10)?g, (0.560.07)?g and (0.560.08)?g, respectively, in 1, 2, 4 and 12 weeks after BTXA injection compared with the normal control group (0.560.08)?g. Changes in the salivary electrolytes and proteins after BTXA injection Amylase concentrations in the rest saliva at 1 and 4 weeks after BTXA administration were 55.2% and 131.3% higher than those in normal control group; however, potassium, sodium and chlorine concentrations remained unchanged. The sodium concentration in the stimulated saliva collected during feeding at 1 and 4 weeks after BTXA treatment increased to 133.7% and 159% of the normal control group ( em P /em 0.05); however, the amylase, potassium, and chlorine concentrations did not change (Number 1c?and?1d). Histological changes after BTXA injection Normal acinar and ductal cells could be observed in the normal control SMGs by light microscopy (Number 2a). No morphological switch was found in the contralateral SMGs with normal saline injection. One week after BTXA injection, the acinar and ductal cells in the glandular lobules at the site of BTXA injection was replaced by fibrous cells. Acinar cells surrounding the injection point showed size reduction, while ducts dilated slightly (Number 2b?and?2c). Four weeks later, the morphological appearance of BTXA-injected SMGs recovered partly. Twelve weeks later on, the treated cells appeared morphologically similar to the normal control glands (Number 2d?and?2e). Open in a separate window Number 2 Histological structure of submandibular glands under 400 magnification. (a) Histological evaluation of a control submandibular gland. (b) Submandubular gland at 1 week after BTXA injection. The normal structure of acinar and ductal cells in the gland lobule at the site of the injection disappeared and was replaced by fibrous cells. (c) Submandubular gland at 2 weeks after BTXA injection. Acinar cells showed a reduction in size, while the ductal cells expanded slightly. (d) Submandubular gland at 4 weeks after BTXA injection. (e) Submandubular gland at 12 weeks after BTXA injection. The morphological appearance of.Pub=2 m. a large ovoid structure. Manifestation of M3-muscarinic acetylcholine receptor (M3 receptor) and aquaporin-5 (AQP5) mRNA decreased after BTXA treatment, and distribution of AQP5 in the apical membrane was reduced at 1, 2 and 4 weeks after BTXA injection. Furthermore, BTXA injection was found to induce apoptosis of acini. These results indicate that BTXA decreases the fluid secretion of submandibular glands and increases the concentration of amylase in saliva. Decreased manifestation of M3 receptor and AQP5, inhibition of AQP5 translocation, and cell apoptosis might involve in BTXA-reduced fluid secretion of submandibular glands. cell-death detection kit (Roche Applied Technology, Penzberg, Germany) was used according to the manufacturer’s instructions. Briefly, tissue sections were incubated with terminal deoxynucleotidyl transferase inside a humidified chamber at 37?C for 1?h. A mixture of antidigoxigenin-peroxidase and substrate-chromagen were utilized for visualization, and the cells were counterstained with hematoxylin. The nuclei of the apoptotic cells were stained dark brown. Reverse transcriptase polymerase chain reaction Total RNA from SMG was purified using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. cDNA was prepared from 5 g of total RNA with Moloney murine leukemia computer virus reverse-transcriptase (Promega, Madison, WI, USA). The sense and antisense primers for M3-muscarinic acetylcholine receptor (M3 receptor) (“type”:”entrez-nucleotide”,”attrs”:”text”:”NM_000740″,”term_id”:”1889688494″NM_000740), AQP5 (AF_495879) and -actin were 5-CTT CTC CAA GCT TCC CAT CCA-3 and 5-TGA CCG ACT GTC TCT GCT GGT-3, 5-GTT CCT GGC CAC CCT CAT CT-3 and 5-ACA GAC AGG CCG ATG GAC AG-3, and 5-ATC TGG CAC CAC ACC TTC TAC AAT GAG CTG GCG-3 and 5-CGC CAT ACT CCT GCT TGC TGA TCC ACA TCT GC-3, respectively.15 -actin was amplified as the internal standard. The amplification products were visualized on 1.5% agarose gel with ethidium bromide and sequenced to confirm their identities. The band densities were quantified using a LEICA-550IW image analysis system (Leica, Manheim, Germany). Immunofluorescence Frozen SMG sections were immunostained with anti-AQP5 antibody at a 1100 dilution, and incubated with FITC- or tetramethylrhodamine isothiocyanate-labeled secondary antibodies as described previously.14 Nuclei were stained RITA (NSC 652287) with 4,6 -diamidino-2-phenylindole (Sigma-Aldrich, St Louis, MO, USA). Fluorescence images were captured under a confocal microscope (TCS SP5; Leica, Heidelberg, Germany). Statistical analysis Data are presented as (means.d.). Differences among groups were analyzed by one-way analysis of variance, and then Bonferroni testing. A value of control. BTXA, botulinum toxin type A; SMG, submandibular gland. There were no significant differences between the weights of SMGs injected with BTXA and the normal control glands. The weights of SMGs was (0.490.08)?g, (0.550.10)?g, (0.560.07)?g and (0.560.08)?g, respectively, in 1, 2, 4 and 12 weeks after BTXA injection compared with the normal control group (0.560.08)?g. Changes in the salivary electrolytes and proteins after BTXA injection Amylase concentrations in the rest saliva at 1 and 4 weeks after BTXA administration were 55.2% and 131.3% higher than those in normal control group; however, potassium, sodium and chlorine concentrations remained unchanged. The sodium concentration in the stimulated saliva collected during feeding at 1 and 4 weeks after BTXA treatment increased to 133.7% and 159% of the normal control group ( em P /em 0.05); however, the amylase, potassium, and chlorine concentrations did not RITA (NSC 652287) change (Physique 1c?and?1d). Histological changes after BTXA injection Normal acinar and ductal cells could be observed in the normal control SMGs by light microscopy (Physique 2a). No morphological change was found in the contralateral SMGs with normal saline injection. One week after BTXA injection, the acinar and ductal cells in the glandular lobules at the site of BTXA injection was replaced by fibrous tissue. Acinar cells surrounding the injection point showed size reduction, while ducts dilated slightly (Physique 2b?and?2c). Four weeks later, the morphological appearance of BTXA-injected SMGs recovered partly. Twelve weeks later, the treated cells appeared morphologically similar to the normal control glands (Physique 2d?and?2e). Open in a separate window Physique 2 Histological structure of submandibular glands under 400 magnification. (a) Histological evaluation of a control submandibular gland. (b) Submandubular gland at 1 week after BTXA injection. The normal structure of acinar and ductal cells in the gland lobule at the site of the injection disappeared and was replaced by fibrous tissue. (c) Submandubular gland at 2 weeks after BTXA injection. Acinar cells showed a reduction in size, while.A mixture of antidigoxigenin-peroxidase and substrate-chromagen were used for visualization, and the cells were counterstained with hematoxylin. of AQP5 translocation, and cell apoptosis might involve in BTXA-reduced fluid secretion of submandibular glands. cell-death detection kit (Roche Applied Science, Penzberg, Germany) was used according to the manufacturer’s instructions. Briefly, tissue sections were incubated with terminal deoxynucleotidyl transferase in a humidified chamber at 37?C for 1?h. A mixture of antidigoxigenin-peroxidase and substrate-chromagen were used for visualization, and the cells were counterstained with hematoxylin. The nuclei of the apoptotic cells were stained dark brown. Reverse transcriptase polymerase chain reaction Total RNA from SMG was purified using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. cDNA was prepared from 5 g of RITA (NSC 652287) total RNA with Moloney murine leukemia computer virus reverse-transcriptase (Promega, Madison, WI, USA). The sense and antisense primers for M3-muscarinic acetylcholine receptor (M3 receptor) (“type”:”entrez-nucleotide”,”attrs”:”text”:”NM_000740″,”term_id”:”1889688494″NM_000740), AQP5 (AF_495879) and -actin were 5-CTT CTC CAA GCT TCC CAT CCA-3 and 5-TGA CCG ACT GTC TCT GCT GGT-3, 5-GTT CCT GGC CAC CCT CAT CT-3 and 5-ACA GAC AGG CCG ATG GAC AG-3, and 5-ATC TGG CAC CAC ACC TTC TAC AAT GAG CTG GCG-3 and 5-CGC CAT ACT CCT GCT TGC TGA TCC ACA TCT GC-3, respectively.15 -actin was amplified as the internal standard. The amplification products were visualized on 1.5% agarose gel with ethidium bromide and sequenced to confirm their identities. The band densities were quantified using a LEICA-550IW image analysis system (Leica, Manheim, Germany). Immunofluorescence Frozen SMG sections were immunostained with anti-AQP5 antibody at a 1100 dilution, and incubated with FITC- or tetramethylrhodamine isothiocyanate-labeled secondary antibodies as described previously.14 Nuclei were stained with 4,6 -diamidino-2-phenylindole (Sigma-Aldrich, St Louis, MO, USA). Fluorescence images were captured under a confocal microscope (TCS SP5; Leica, Heidelberg, Germany). Statistical analysis Data are presented as (means.d.). Differences among groups were analyzed by one-way analysis of variance, and then Bonferroni testing. A value of control. BTXA, botulinum toxin type A; SMG, submandibular gland. There were no significant differences between the weights of SMGs injected with BTXA and the normal control glands. The weights of SMGs was (0.490.08)?g, (0.550.10)?g, (0.560.07)?g and (0.560.08)?g, respectively, in 1, 2, 4 and 12 weeks after BTXA injection compared with the normal control group (0.560.08)?g. Changes in the salivary electrolytes and proteins after BTXA injection Amylase concentrations in the rest saliva at 1 and 4 weeks after BTXA administration were 55.2% and 131.3% higher than those in normal control group; however, potassium, sodium and chlorine concentrations remained unchanged. The sodium concentration in the stimulated saliva collected during feeding at 1 and 4 weeks after BTXA treatment increased to 133.7% and 159% of the normal control group ( em P /em 0.05); however, the amylase, potassium, and chlorine concentrations did not change (Physique 1c?and?1d). Histological changes after BTXA injection Normal acinar and ductal cells could be observed in the standard control SMGs by light microscopy (Shape 2a). No morphological modification was within the contralateral SMGs with regular saline shot. Seven days after BTXA shot, the acinar and ductal cells in the glandular lobules at the website of BTXA shot was changed by fibrous cells. Acinar cells encircling the shot point demonstrated size decrease, while ducts dilated somewhat (Shape 2b?and?2c). A month later on, the morphological appearance of BTXA-injected SMGs retrieved partially. Twelve weeks later on, the treated cells made an appearance morphologically like the regular control glands (Shape 2d?and?2e). Open up in another window Shape 2 Histological framework of submandibular glands under 400 magnification. (a) Histological evaluation of the control submandibular gland. (b) Submandubular gland at a week after BTXA shot. The normal framework of acinar and ductal cells in the gland lobule at the website of the shot vanished and was changed by fibrous cells. (c) Submandubular gland at 14 days after BTXA shot. Acinar cells demonstrated a decrease in size, as the ductal cells extended somewhat. (d) Submandubular gland at four weeks after BTXA shot. (e) Submandubular gland at 12 weeks after BTXA shot. The morphological appearance of acinar cells retrieved to the standard. Pub=50 m. BTXA, botulinum toxin type A. Transmitting electron microscopy exposed that secretory granules of low matrix denseness spread broadly in the cytoplasm of acinar cells in the standard control (Shape 3a), while abnormal.Fluorescence pictures were captured under a confocal microscope (TCS SP5; Leica, Heidelberg, Germany). Statistical analysis Data are presented while (means.d.). decreased at 1, 2 and four weeks after BTXA shot. Furthermore, BTXA shot was discovered to induce apoptosis of acini. These outcomes indicate that BTXA reduces the liquid secretion of submandibular glands and escalates the focus of amylase in saliva. Reduced manifestation of M3 receptor and AQP5, inhibition of AQP5 translocation, and cell apoptosis might involve in BTXA-reduced liquid secretion of submandibular glands. cell-death recognition package (Roche Applied Technology, Penzberg, Germany) was utilized based on the manufacturer’s guidelines. Briefly, tissue areas had been incubated with terminal deoxynucleotidyl transferase inside a humidified chamber at 37?C for 1?h. An assortment of antidigoxigenin-peroxidase and substrate-chromagen were useful for visualization, as well as the cells were counterstained with hematoxylin. The nuclei from the apoptotic cells had been stained darkish. Change transcriptase polymerase string response Total RNA from SMG was purified using Trizol reagent (Invitrogen, Carlsbad, CA, USA) based on the manufacturer’s guidelines. cDNA was ready from 5 g of total RNA with Moloney murine leukemia disease reverse-transcriptase (Promega, Madison, WI, USA). The sense and antisense primers for M3-muscarinic acetylcholine receptor (M3 receptor) (“type”:”entrez-nucleotide”,”attrs”:”text”:”NM_000740″,”term_id”:”1889688494″NM_000740), AQP5 (AF_495879) and -actin had been 5-CTT CTC CAA GDF5 GCT TCC CAT CCA-3 and 5-TGA CCG Work GTC TCT GCT GGT-3, 5-GTT CCT GGC CAC CCT CAT CT-3 and 5-ACA GAC AGG CCG ATG GAC AG-3, and 5-ATC TGG CAC CAC ACC TTC TAC AAT GAG CTG GCG-3 and 5-CGC CAT Work CCT GCT TGC TGA TCC ACA TCT GC-3, respectively.15 -actin was amplified as the inner standard. The amplification items had been visualized on 1.5% agarose gel with ethidium bromide and sequenced to verify their identities. The music group densities had been quantified utilizing a LEICA-550IW picture analysis program (Leica, Manheim, Germany). Immunofluorescence Frozen SMG areas had been immunostained with anti-AQP5 antibody at a 1100 dilution, and incubated with FITC- or tetramethylrhodamine isothiocyanate-labeled supplementary antibodies as referred to previously.14 Nuclei were stained with 4,6 -diamidino-2-phenylindole (Sigma-Aldrich, St Louis, MO, USA). Fluorescence pictures had been captured under a confocal microscope (TCS SP5; Leica, Heidelberg, Germany). Statistical evaluation Data are shown as (means.d.). Variations among groups had been examined by one-way evaluation of variance, and Bonferroni tests. A worth of control. BTXA, botulinum toxin type A; SMG, submandibular gland. There have been no significant variations between your weights of SMGs injected with BTXA and the standard control glands. The weights of SMGs was (0.490.08)?g, (0.550.10)?g, (0.560.07)?g and (0.560.08)?g, respectively, in 1, 2, 4 and 12 weeks after BTXA shot compared with the standard control group (0.560.08)?g. Adjustments in the salivary electrolytes and protein after BTXA shot Amylase concentrations in the others saliva at 1 and four weeks after BTXA administration had been 55.2% and 131.3% greater than those in normal control group; nevertheless, potassium, sodium and chlorine concentrations continued to be unchanged. The sodium focus in the activated saliva gathered during nourishing at 1 and four weeks after BTXA treatment risen to 133.7% and 159% of the standard control group ( em P /em 0.05); nevertheless, the amylase, potassium, and chlorine concentrations didn’t change (Shape 1c?and?1d). Histological adjustments after BTXA shot Regular acinar and ductal cells could possibly be observed in the standard control SMGs by light microscopy (Shape 2a). No morphological modification was within the contralateral SMGs with regular saline shot. Seven days after BTXA shot, the acinar and ductal cells in the glandular lobules at the website of BTXA shot was changed by fibrous cells. Acinar cells encircling the shot point demonstrated size decrease, while ducts dilated somewhat (Shape 2b?and?2c). A month later on, the morphological appearance of BTXA-injected SMGs retrieved partially. Twelve weeks later on, the treated cells made an appearance morphologically like the regular control glands (Shape 2d?and?2e). Open up in another window Shape 2 Histological framework of submandibular glands under 400 magnification. (a) Histological evaluation of the control submandibular gland. (b) Submandubular gland at a week after BTXA shot. The normal framework of acinar and ductal cells in the gland lobule at the website of the shot vanished and was changed by fibrous cells. (c) Submandubular gland at 14 days after BTXA shot. Acinar cells demonstrated a decrease in size, as the ductal cells extended somewhat. (d) Submandubular gland at four weeks after BTXA shot. (e) Submandubular gland at 12 weeks after BTXA shot. The morphological appearance of acinar cells retrieved to the standard. Pub=50 m. BTXA, botulinum toxin type A. Transmitting electron microscopy exposed that secretory granules of low.