Types of cell culture inserts affect cell crosstalk between co-cultured macrophages and adipocytes.

Cell culture inserts offer an in vivo-like microenvironment to investigate cell-cell interactions between co-cultivated cells. However, it is unclear if types of inserts affect cell crosstalk. Here, we developed an environment-friendly cell culture insert, XL-insert, which can reduce plastic waste with lower cost. We compared XL insert with two types of commercial disposable culture inserts, Koken® insert with atelocollagen membrane (Col-inserts) and Falcon® inserts with plastic membrane (PET-inserts) on cell-cell interactions in co-cultivated THP-1 macrophages and OP9 adipocytes. Scanning electron microscope, immunoassay and imaging analysis showed that among three types of inserts, XL-inserts allowed cytokines from co-cultivated macrophages and adipocytes to diffuse freely and offered preferable in vivo-like microenvironment for cell-cell interactions. PET-inserts showed limitations for intercellular communication due to some pores being blocked by somas on the membrane that caused much lower permeability for cytokines passing through. Col-inserts blocked large sized cytokines but allowed small sized molecules to permeate resulting in improved lipid accumulation and adiponectin secretion in OP9 adipocytes. Taken together, our data demonstrated that membrane type and pore size on the membrane affect the cross-talk between co-cultivated cells very differently. Some previous co-culture studies might have different results if the inserts were changed.

Enzyme-Digested Edible Bird's Nest (EBND) Prevents UV and Arid Environment-Induced Cellular Oxidative Stress, Cell Death and DNA Damage in Human Skin Keratinocytes and Three-Dimensional Epithelium Equivalents.

The aim of this study is to investigate the repressive effects of enzyme-digested edible bird's nest (EBND) on the combination of arid environment and UV-induced intracellular oxidative stress, cell death, DNA double-strand breaks (DSBs) and inflammatory responses in human HaCaT keratinocytes and three-dimensional (3D) epithelium equivalents. An oxygen radical antioxidant capacity assay showed that EBND exhibited excellent peroxyl radical scavenging activity and significantly increased cellular antioxidant capacity in HaCaT cells. When EBND was administered to HaCaT cells and 3D epitheliums, it exhibited significant preventive effects on air-drying and UVA (Dry-UVA)-induced cell death and apoptosis. Dry-UVA markedly induced intracellular reactive oxygen species (ROS) generation in HaCaT cells and 3D epitheliums as quantified by CellROX® Green/Orange reagents. Once HaCaT cells and 3D epitheliums were pretreated with EBND, Dry-UVA-induced intracellular ROS were significantly reduced. The results from anti-γ-H2A.X antibody-based immunostaining showed that EBND significantly inhibited Dry-UVA-induced DSBs in HaCaT keratinocytes. Compared with sialic acid, EBND showed significantly better protection for both keratinocytes and 3D epitheliums against Dry-UVA-induced injuries. ELISA showed that EBND significantly suppressed UVB-induced IL-6 and TNF-α secretion. In conclusion, EBND could decrease arid environments and UV-induced harmful effects and inflammatory responses in human keratinocytes and 3D epithelium equivalents partially through its antioxidant capacity.

Buccal Nerve Trunk Block Anesthetizes the Buccal Mucosa Beyond the Papilla of the Parotid Duct.

PURPOSE: The limited area of anesthesia of the buccal mucosa with concomitant conventional buccal nerve block (conventional BNB) may be involved in failed inferior alveolar nerve block (IANB). The aims of this study were to examine the extent of anesthesia by buccal nerve trunk block (BNTB) and compare the success rates of IANB with BNB. METHODS: This prospective parallel-group randomized single-blinded clinical trial included patients scheduled for removal of a mandibular third molar at the Nippon Dental University Hospital between September 2021 and March 2022. The primary predictor variable was the approach for BNB (BNTB vs conventional BNB). The primary outcome was the extent of tactile sensory loss and anesthesia of the buccal mucosa. The secondary outcomes included onset time and duration of BNBs and the success rate of the IANB with concomitant BNB for third molar extraction, assessed by the proportions of intraoperative pain perception and supplemental infiltration anesthesia. The other study variables were sex, age, and injection side. Comparisons were analyzed by Fisher's exact test or the Mann-Whitney U test. The P value was set to .05. RESULTS: A total of 38 patients (14 male, 24 female) with a mean age of 28.9 years (range, 18 to 67 years) were enrolled, with 19 patients each in the BNTB group and conventional BNB group. The effective tactile sensory loss rates at ∼5 mm above the height of the papilla of the parotid duct of the premolar and molar regions were greater following BNTB (71 and 95%, respectively) than following conventional BNB (37%; P < .01 and 58%; P < .01, respectively). The proportions of intraoperative pain perception of the BNTB group and the conventional BNB group were 10 and 42% (P = .06), respectively, and those of supplemental infiltration anesthesia were 5 and 26% (P = .18), respectively. CONCLUSIONS: BNTB provided a wider extent of anesthesia of the buccal mucosa than conventional BNB and may improve the anesthesia success of IANB for removal of mandibular third molars.

Usefulness of preoperative computed tomography findings for airway management in patients with acute odontogenic infection: a retrospective study.

Odontogenic infection is more likely to affect the airway and interfere with intubation than non-odontogenic causes. Although anesthesiologists predict the difficulty of intubation and determine the method, they may encounter unexpected cases of difficult intubation. An inappropriate intubation can cause airway obstruction due to bleeding and edema by damaging the pharynx and larynx. This study was performed to determine the most important imaging findings indicating preoperative selection of an appropriate intubation method. This retrospective study included 113 patients who underwent anti-inflammatory treatment for odontogenic infection. The patients were divided into two groups according to the intubation method: a Macintosh laryngoscope (45 patients) and others (video laryngoscope and fiberscope) (68 patients). The extent of inflammation in each causative tooth, the severity of inflammation (S1-4), and their influence on the airway were evaluated by computed tomography. The causative teeth were mandibular molars in more than 90%. As the severity of inflammation increased, anesthesiologists tended to choose intubation methods other than Macintosh laryngoscopy. In the most severe cases (S4), anesthesiologists significantly preferred other intubation methods (33 cases) over Macintosh laryngoscopy (9 cases). All patients with S4 showed inflammation in the parapharyngeal space, and the airway was affected in 41 patients. The mandibular molars were the causative teeth most likely to affect the airway and surrounding region. In addition to clinical findings, the presence or absence of inflammation that has spread to the parapharyngeal space on preoperative computed tomography was considered an important indicator of the difficulty of intubation.

Anesthetic Management of a Patient With Catecholaminergic Polymorphic Ventricular Tachycardia.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a rare inherited arrhythmogenic disorder induced by adrenergic stress. Electrophysiologically, it is characterized by emotional stress- or exercise-induced bidirectional ventricular tachycardia that may result in cardiac arrest. Minimizing perioperative stress is critical as it can reduce fatal arrhythmias in patients with CPVT. Dexmedetomidine (DEX), a centrally acting sympatholytic anesthetic agent, was used in the successful intravenous (IV) moderate sedation of a 27-year-old female patient with CPVT, a history of cardiac events, and significant dental fear and anxiety scheduled to undergo mandibular left third molar extraction. Oral surgery was successfully performed under DEX-based IV sedation to reduce stress, and no arrhythmias were observed. IV sedation with DEX provided a sympatholytic effect with respiratory and cardiovascular stability in this patient with CPVT who underwent oral surgery.

Effects of vasopressin administration in the oral cavity on cardiac function and hemodynamics in rats.

BACKGROUND: The vasoconstrictive effect of epinephrine in local anesthetics affects the heart, which leads to hesitation among dentists in injecting local anesthetics into patients with cardiovascular disease. Due to its vasoconstrictive effects, the present study investigated the effects of vasopressin administration on cardiac function in rats. METHODS: Experiment 1 aimed to determine the vasopressin concentration that could affect cardiac function. An arterial catheter was inserted into the male Wistar rats. Next, 0.03, 0.3, and 3.0 U/mL arginine vasopressin (AVP) (0.03V, 0.3V, and 3.0V) was injected into the tongue, and the blood pressure was measured. The control group received normal saline only. In Experiment 2, following anesthesia infiltration, a pressure-volume catheter was placed in the left ventricle. Baseline values of end-systolic elastance, end-diastolic volume, end-systolic pressure, stroke work, stroke volume, and end-systolic elastance were recorded. Next, normal saline and 3.0V AVP were injected into the tongue to measure their effect on hemodynamic and cardiac function. RESULTS: After 3.0V administration, systolic blood pressures at 10 and 15 min were higher than those of the control group; they increased at 10 min compared with those at baseline. The diastolic blood pressures at 5-15 min were higher than those of the control group; they increased at 5 and 10 min compared with those at baseline. The preload decreased at 5 and 10 min compared to that at baseline. However, the afterload increased from 5 to 15 min compared with that of the control group; it increased at 10 min compared with that at baseline. Stroke volume decreased at 10 and 15 min compared with that of the control group; it decreased from 5 to 15 min compared with that at baseline. Stroke work decreased from 5 to 15 min compared with that of the control group; it decreased from 5 to 15 min compared with that at baseline. CONCLUSION: Our results showed that 3.0 U/mL concentration of vasopressin resulted in increased blood pressure, decreased stroke volume and stoke work, decreased preload and increased afterload, without any effect on myocardial contractility.

Hemodynamic Impact of Drug Interactions With Epinephrine and Antipsychotics Under General Anesthesia With Propofol.

OBJECTIVE: Antipsychotic drugs exhibit α-1 adrenergic receptor-blocking activity. When epinephrine and antipsychotic drugs are administered in combination, ß-2 adrenergic effects are thought to predominate and induce hypotension. This study aimed to assess hemodynamic parameters in patients regularly taking antipsychotics who were administered epinephrine-containing lidocaine under general anesthesia in a dental setting. METHODS: Thirty patients taking typical and/or atypical antipsychotics and scheduled for dental procedures under general anesthesia were enrolled. Five minutes after tracheal intubation, baseline systolic blood pressure (SBP), diastolic blood pressure (DBP), heart rate (HR), and percutaneous oxygen saturation (SpO2) measurements were taken. The SBP, DBP, HR, and SpO2 measurements were repeated 2, 4, 6, 8, and 10 minutes after the injection of 1.8 mL of 2% lidocaine (36 mg) with 1:80,000 epinephrine (22.5 mcg) via buccal infiltration. RESULTS: Differences between the baseline measurements and those of each time point were analyzed using Dunnett test, and no statistically significant changes were observed. CONCLUSIONS: Our findings demonstrate that the use of epinephrine at a clinically relevant dose of 22.5 mcg for dental treatment under general anesthesia is unlikely to affect the hemodynamic parameters of patients taking antipsychotic medications.

Effect of epinephrine on the absorption of lidocaine following application to the oral mucosa in rats.

BACKGROUND: We investigated the role of epinephrine in prolonging the localization of lidocaine on the oral mucosa and inhibiting its absorption in the blood of rats. METHODS: We used 7-8-week-old pathogen-free Wistar male rats (n = 128) for our study. We divided them into the control group administered with 14C-labeled lidocaine hydrochloride gel only and the study group administered with 14C-labeled lidocaine hydrochloride gel with epinephrine. The medications were administered in the palatal mucosa of the rats. The amount of mucosa, palatine bone, and serum lidocaine was measured by radioactivity using a liquid scintillation counter and was observed using autoradiograms. RESULTS: Initially, there was no significant difference in the lidocaine levels between the lidocaine and lidocaine with epinephrine groups in the palatal mucosa (751.9 ± 133.8 vs. 669.8 ± 101.6 ng/mg [2 min]). After 4 min, the values were significantly lower in the lidocaine with epinephrine group (1040.0 ± 142.8 vs. 701.2 ± 109.0 ng/mg [20 min]). After 40 min, the lidocaine level became significantly higher in the lidocaine with epinephrine group (586.8 ± 112.4 vs. 1131.3 ± 155.2 ng/mg [40 min]). Similar results were observed in the palatine bone and serum. CONCLUSION: Epinephrine prolonged the localization of lidocaine applied to the mucosa and inhibited its absorption into the bloodstream of rats. Clinical studies are required to evaluate the use of epinephrine-containing topical anesthetics on the oral mucosa.

Effects of vasopressin on anesthetic response time and circulatory dynamics of lidocaine.

This study aimed to investigate the hypothesis that vasopressin extends the anesthetic response time of lidocaine and does not affect the circulatory dynamics. Rats were sedated with isoflurane; subsequently, breathing was maintained through mechanical ventilation. We infiltrated the first molar area of the upper left jaw with saline (NS, test solution), 2% lidocaine (L), 0.025 IU vasopressin-supplemented 2% lidocaine, 0.05 IU vasopressin-supplemented 2% lidocaine, 0.1 IU vasopressin-supplemented 2% lidocaine, and 0.2 IU vasopressin-supplemented 2% lidocaine (VL4). Further, anesthetic response times were measured up to 30 min using electric pulp testing methods (n = 4). The anesthetic response times of NS, L, and VL4 were measured up to 45 min with the aforementioned results as reference values (n = 7). The circulatory dynamics of NS, L, VL4, and 0.2 IU vasopressin (V) were measured up to 45 min using a non-invasive blood pressure measuring device. VL4 extended the anesthetic response times of lidocaine compared to L (p < 0.05). Further, V and VL4 significantly increased the systolic and diastolic blood pressure and significantly decreased the pulse rate (p < 0.05). VL4 is not a suitable addition to the local anesthetic solution used in dentistry. Further study is needed to determine vasopressin concentration that extends the anesthetic effect without affecting the circulatory dynamics.

Effect of epinephrine on the distribution of ropivacaine and lidocaine using radioactive isotopes in rat maxilla and pulp.

We compared the effect of epinephrine on the distribution of ropivacaine and lidocaine by using radioactive isotopes in rat maxilla and pulp. Twenty microliters of 3H-labeled 0.5% ropivacaine, 14C-labeled 2.0% lidocaine, or epinephrine-supplemented isotopes were injected into the maxilla. The radioactivity was measured and autoradiography was obtained. Epinephrine led to increase in amounts of both anesthetics in the maxilla and pulp; however, each anesthetic did so in a different manner. Addition of epinephrine to lidocaine decreased radioactivity in maxilla and pulp with time. Conversely, when ropivacaine with epinephrine was administered, radioactivity did not change until 20 min in the maxilla and reached its peak at 20 min in the pulp. Autoradiography of lidocaine faded with time even with epinephrine use; however, with ropivacaine, higher accumulation image was observed after 20 min compared to that after 2 min. When epinephrine was combined with lidocaine, the amount of lidocaine in maxilla and pulp decreased with time, similar to when lidocaine was used alone. Conversely, when ropivacaine-epinephrine combination was administered, the amount of ropivacaine remained unchanged for 20 min in the maxilla and reached its peak at 20 min in the dental pulp.

Adding Dexmedetomidine to Articaine Increases the Latency of Thermal Antinociception in Rats.

Articaine is a low-toxicity local anesthetic that is widely used in dentistry. Typically, epinephrine is added to prolong the duration of articaine local anesthesia; however, epinephrine exhibits adverse effects. Low-dose dexmedetomidine (DEX), an α2-adrenoreceptor agonist, reportedly prolongs local anesthesia without notable adverse cardiovascular effects. The purpose of this study was to assess whether a combination of low-dose DEX and articaine would provide a low-toxicity local anesthetic option for dental procedures without adverse cardiovascular effects. Thus, this study investigated whether DEX could prolong the local anesthetic effect of articaine using a rat model of pain. Adult male Wistar rats (N = 44; 11 per group) received a 50-µL subcutaneous injection into the plantar surface of the hind paws; injections were composed of either normal saline, 4% articaine (2 mg articaine), combined 5 µg/kg DEX and 4% articaine (1.25 µg DEX + 2 mg articaine), or combined epinephrine (1:100,000) and 4% articaine (0.9 µg epinephrine + 2 mg articaine). Subsequent acute pain perception was determined by paw withdrawal movement in response to infrared radiant heat stimulation of the plantar region. Paw withdrawal latency was tested at 5-minute intervals. Paw withdrawal latency values at 35 and 40 minutes were 3.83 ± 1.76 and 3.29 ± 1.43 seconds for articaine alone, 7.89 ± 2.72 and 7.25 ± 3.37 seconds for DEX and articaine, and 8.95 ± 2.28 and 8.17 ± 3.01 seconds for epinephrine and articaine. DEX prolonged the paw withdrawal latency of articaine for up to 35 minutes (p = .015) but not 40 minutes after injection (p = .052) when compared to articaine alone. The combination of DEX and articaine can provide effective local anesthesia for up to 35 minutes after injection.

Effect of lidocaine with adrenaline and landiolol on hemodynamics in spontaneously hypertensive rats.

Administration of local anesthetics with adrenaline can cause tachycardia and hypertension. This study assessed whether combined administration of landiolol with adrenaline and lidocaine would induce local anesthesia without causing hemodynamic changes. Normal saline (NS), lidocaine with adrenaline (LA), and lidocaine with adrenaline and landiolol (LLA) were injected into Wistar Kyoto (WKY/Izm) or spontaneously hypertensive (SHR/Izm) rats, followed by measurement of the pulse rate (PR), and the systolic, diastolic and mean blood pressures (SBP, DBP and MBP). In the LLA group, the increase in PR was significantly suppressed in both SHR/Izm and WKY/Izm rats relative to those in the LA group. Although SBP was significantly reduced in WKY/Izm rats given LLA, relative to those given NS or LA, it was elevated in SHR/Izm rats given LLA. Landiolol-induced changes in PR may be due to blockade of adrenaline-induced ß1 receptor stimulation, which suppresses cardiac hyperactivity, whereas the early surge of blood pressure in SHR/Izm rats given LLA may be due to the dominant alpha-adrenergic effects of ß1 receptor inhibition. The anti-adrenergic effects of LLA were safe and effective in WKY/Izm rats, although the unexpected early hypertensive surge in SHR/Izm rats indicates the need for caution.

Anesthetic Management of a Patient With Systemic Sclerosis and Microstomia.

Systemic sclerosis (SSc) is an autoimmune disease that can cause fibrosis in vital organs, often resulting in damage to the skin, blood vessels, gastrointestinal system, lungs, heart, and/or kidneys. Patients with SSc are also likely to develop microstomia, which can render dental treatment difficult and painful, thereby necessitating advanced anesthetic management. This is a case report of a 61-year-old woman with a history of SSc with microstomia, interstitial pneumonia, and gastroesophageal reflux disease in whom intravenous moderate sedation was performed using a combination of dexmedetomidine and ketamine for dental extractions. Both anesthetic agents are known to have analgesic effects while minimizing respiratory depression. Consequently, the increased discomfort caused by opening the patient's mouth and stretching the buccal mucosa was sufficiently managed, permitting an increase in maximum interincisal opening and completion of treatment without complications. Patients with SSc present with serious comorbidities that can negatively impact anesthetic management, so the implementation of an anesthetic plan that takes such risks into account is required. Furthermore, emergency airway management is likely to be difficult in patients with microstomia. For intravenous moderate sedation, combined use of dexmedetomidine and ketamine, which have analgesic effects while minimizing respiratory depression, may be particularly effective in patients with SSc and microstomia.

Anesthetic Management of a Pediatric Patient With Cardiofaciocutaneous Syndrome.

Cardiofaciocutaneous (CFC) syndrome is a rare condition characterized by congenital heart disease, craniofacial dysmorphology, and dermatological abnormalities. CFC syndrome is one of the RASopathies, a family of syndromes that also includes Noonan and Costello syndromes, all with underlying gene mutations involving the Ras/mitogen-activated protein kinase pathways. Important considerations for anesthesiologists caring for these patients include the need to evaluate for possible cardiac defects, anticipating and planning for potentially difficult airway management, and the consideration of potential weakness of the respiratory muscles. Musculoskeletal abnormalities, such as muscle weakness and decreased muscle mass, are observed in all RASopathies, but are particularly prominent in CFC syndrome. In patients with CFC syndrome who experience respiratory muscle weakness, the use of desflurane and remifentanil may aid in a faster recovery and effectively help reduce the risk of respiratory complications, such as respiratory depression, following general anesthesia because of their rapid metabolism or elimination.

Effect of adding vasopressin on the distribution of lidocaine in tissues, anesthetic action, and circulatory dynamics.

We examined whether vasopressin affects the distribution, anesthesia duration, and circulatory dynamics of lidocaine. Blood flow was measured after injecting 0.003, 0.03, or 0.3 U/mL vasopressin and 2% lidocaine (L) to the upper lip of rats. Radioactivity and distribution of 14C-labeled L (CL) in the palate, palatal mucosa, maxilla bone, and blood was measured by autoradiography after injecting CL and CL + 0.03 U/mL vasopressin. To evaluate anesthesia duration, somatosensory-evoked potentials, blood pressure, and pulse rate were measured after L, 0.03 U/mL vasopressin, and L + 0.03 U/mL vasopressin injection to the palatal mucosa. Blood flow from 10 to 60 min was significantly lower with 0.03 U/mL vasopressin and L + 0.03 U/mL vasopressin than with L. Radioactivity in the palatal mucosa and maxilla bone was significantly higher at 5-60 min and 2-60 min with CL + 0.03 U/mL vasopressin than with CL. Blood radioactivity reached the maximum at 0.5 and 50 min with CL and CL + 0.03 U/mL vasopressin, respectively. Autoradiogram showed higher distribution with CL + 0.03 U/mL vasopressin than CL. Peak-to-peak amplitude 30-60 min was significantly lower with L + 0.03 U/mL vasopressin than with L. Lidocaine did not affect blood pressure and pulse rate with 0.03 U/mL vasopressin-only or combined with 2%-lidocaine. Topical 0.03 U/mL vasopressin injection reduced the tissue blood flow, promoted the localization and retention, and extended the anesthesia duration of lidocaine, leaving circulatory dynamics unaffected.

Anesthetic management in corticobasal degeneration with central sleep apnea: A case report.

Corticobasal degeneration (CBD) is a rare neurodegenerative disease characterized by dystonia, cognitive deficits, and an asymmetric akinetic-rigid syndrome. Little information is available regarding anesthetic management for CBD patients. Our patient was a 55-year-old man with CBD complicated by central sleep apnea (CSA). Due to the risk of perioperative breathing instability associated with anesthetic use, a laryngeal mask airway was used during anesthesia with propofol. Spontaneous respiration was stable under general anesthesia. However, respiratory depression occurred following surgery, necessitating insertion of a nasopharyngeal airway. Since no respiratory depression had occurred during maintenance of the airway using the laryngeal mask, we suspected an upper airway obstruction caused by displacement of the tongue due to residual propofol. Residual anesthetics may cause postoperative respiratory depression in patients with CBD. Therefore, continuous postoperative monitoring of SpO2 and preparations to support postoperative ventilation are necessary.

General Anesthesia in a Glucose-6-Phosphate Dehydrogenase Deficiency Child: A Case Report.

We performed general anesthesia on a 3-year-old boy with glucose-6-phosphate dehydrogenase (G6PD) deficiency. Patients with G6PD deficiency exhibit jaundice and anemia due to hemolysis caused by a lack of the G6PD enzyme. To maintain anesthesia, we used propofol and remifentanil, which may prevent hemolytic attacks by exerting an antioxidant effect. In addition, because the patient was in a high-risk group for the development of methemoglobinemia, we used mepivacaine as a local anesthetic. We liaised with the patient's attending physician to make sufficient arrangements, such as securing an emergency transfer on the day of anesthesia. The patient did not develop hemolytic attacks during or after the procedure, and he progressed well without problems.

Intravenous Sedation in Arnold-Chiari Malformation With Respiratory Failure.

Type II Arnold-Chiari malformation (ACM) is an abnormality in which the cerebellum, pons, and medulla oblongata are displaced downward into the spinal cord. Type II ACM is often complicated by respiratory depression, sleep-disordered breathing, and deglutition disorder as a result of medullary dysfunction and impairment of the lower cranial nerves. Bending and stretching of the neck is restricted, and anesthetic management is problematic in patients with the disorder. We performed dental treatment twice under intravenous sedation in a patient with intellectual disability with type II ACM complicated by hypercapnic respiratory failure. Propofol was used for the first sedation procedure. Repeated bouts of respiratory depression occurred on that occasion, so the airway was managed manually by lifting the jaw. However, aspiration pneumonitis occurred postoperatively. A combination of dexmedetomidine and midazolam was used for sedation on the second occasion, and the intervention was completed uneventfully without any respiratory depression. Our experience with this patient highlights the need for selection of an agent for intravenous sedation that does not require neck extension and has minimal effect on respiration in patients with type II ACM, who are at high risk of respiratory depression and pulmonary aspiration.

Development and use of a mouth gag for oral experiments in rats.

OBJECTIVE: Rodent models such as mice and rats are often used in investigations of the oral cavity in the fields of periodontology and dental anesthesiology. When various treatments are performed in the oral cavity, it is very important to secure the visual field while keeping the animal's mouth fully opened, in order to ensure the consistency of experimental procedures. Therefore, we developed a standardized gag conforming to various degrees of oral cavity size of different aged rats. DESIGN: The gag was composed of a rectangular incisor-opening frame constructed from a stainless steel wire with retractors and a dial to alter the opening amounts. Wistar rats (n = 5) aged 4, 8, and 12 weeks were used to evaluate the suitability of the gag in oral cavity. As tests for application of gag in intraoral experiments, the ligature placement around the molars, drug injection into the gingiva, measurement of gingival blood flow rate, and installation of stimulation an electrode for somatosensory-evoked potentials into the molar were performed. RESULTS: Adjusting the opening dial enabled both the maintenance of open state and more favorable intraoral observation compared with tweezers as a control device in all different types of rats. Furthermore, our gag made it possible to facilitate the insertion of diverse instruments into the oral cavity and to achieve various experimental purposes. The stainless-steel gag can also be autoclaved and dry-heat sterilized. CONCLUSION: It was revealed that our mouth gag can be widely applied to various oral experiments in different old aged rats.