Prenatal asfotase alfa-mediated enzyme replacement therapy restores delayed calcification in a severe infantile form of hypophosphatasia model mice.

Hypophosphatasia (HPP) is a congenital disorder caused by mutations in the tissue-nonspecific alkaline phosphatase (TNALP) gene. The pathogenesis of HPP varies, ranging from severe cases in which there is total absence of fetal bone calcification, which leads to stillbirth, to relatively mild cases in which the effects are confined to the teeth, such as early loss of the primary teeth. In recent years, the establishment of enzyme supplementation as a treatment method has prolonged survival in patients; however, this approach does not provide sufficient improvement for failed calcification. Furthermore, the effects of enzyme replacement therapy on the jawbone and periodontal tissues have not yet been studied in detail. Therefore, in this study, we investigated the therapeutic effects of enzyme replacement therapy on jawbone hypocalcification in mice. Recombinant TNALP was administered to mothers before birth and newborns immediately after birth, and the effect of treatment was evaluated at 20 days of age. The treated HPP mice had improved mandible (mandibular length and bone quality) and tooth quality (root length of mandibular first molar, formation of cementum), as well as improved periodontal tissue structure (structure of periodontal ligament). Furthermore, prenatal treatment had an additional therapeutic effect on the degree of mandible and enamel calcification. These results suggest that enzyme replacement therapy is effective for the treatment of HPP, specifically in the maxillofacial region (including the teeth and mandible), and that early initiation of treatment may have additional beneficial therapeutic effects.

Generation of bicistronic Dmp1-Cre knock-in mice using a self-cleaving 2A peptide.

INTRODUCTION: The conditional manipulation of genes using the Cre recombinase-locus of crossover in P1 (Cre/loxP) system is an important tool for revealing gene functions and cell lineages in vivo. The outcome of this method is dependent on the performance of Cre-driver mouse strains. In most cases, Cre knock-in mice show better specificity than randomly inserted Cre transgenic mice. However, following knock-in, the expression of the original gene replaced by Cre is lost. MATERIALS AND METHODS: We generated a new differentiated osteoblast- and osteocyte-specific Cre knock-in mouse line that carries the viral T2A sequence encoding a 2A self-cleaving peptide at the end of the coding region of the dentin matrix protein 1 (Dmp1) gene accompanied by the Cre gene. RESULTS: We confirmed that Dmp1-T2A-Cre mice showed high Cre expression in osteoblasts, osteocytes, odontoblasts, and periodontal ligament cells and that the 2A self-cleaving peptide efficiently produced both Dmp1 and Cre proteins. Furthermore, unlike the Dmp1 knockout mice, homozygous Dmp1-T2A-Cre mice showed no skeletal abnormalities. Analysis using the Cre reporter strain confirmed differentiated osteoblast- and osteocyte-specific Cre-mediated recombination in the skeleton. Furthermore, recombination was also detected in some nuclei of skeletal muscle cells, spermatocytes, and intestinal cells. CONCLUSION: 2A-Cre functions effectively in vivo, and Dmp1-T2A-Cre knock-in mice are a useful tool for studying the functioning of various genes in hard tissues.

Subset of the periodontal ligament expressed leptin receptor contributes to part of hard tissue-forming cells.

The lineage of periodontal ligament (PDL) stem cells contributes to alveolar bone (AB) and cementum formation, which are essential for tooth-jawbone attachment. Leptin receptor (LepR), a skeletal stem cell marker, is expressed in PDL; however, the stem cell capacity of LepR+ PDL cells remains unclear. We used a Cre/LoxP-based approach and detected LepR-cre-labeled cells in the perivascular around the root apex; their number increased with age. In the juvenile stage, LepR+ PDL cells differentiated into AB-embedded osteocytes rather than cementocytes, but their contribution to both increased with age. The frequency of LepR+ PDL cell-derived lineages in hard tissue was < 20% per total cells at 1-year-old. Similarly, LepR+ PDL cells differentiated into osteocytes following tooth extraction, but their frequency was < 9%. Additionally, both LepR+ and LepR- PDL cells demonstrated spheroid-forming capacity, which is an indicator of self-renewal. These results indicate that both LepR+ and LepR- PDL populations contributed to hard tissue formation. LepR- PDL cells increased the expression of LepR during spheroid formation, suggesting that the LepR- PDL cells may hierarchically sit upstream of LepR+ PDL cells. Collectively, the origin of hard tissue-forming cells in the PDL is heterogeneous, some of which express LepR.

Effectively improved 3-dimensional structural stability of atelocollagen-gelatin sponge biomaterial by heat treatment.

Atelocollagen-gelatin (ACG) sponge was fabricated from atelocollagen and gelatin by lyophilization without introducing toxic substances. This study aimed to investigate the effects of heat treatment on the 3-dimensional structural stability of ACG sponge biomaterial. ACG sponge samples were fabricated and heat treated at 125oC for 12 h in the vacuum. The results revealed that heat treatment did not affect porosity, pore size and mechanical compressive strength. Heat-treated ACG sponge showed decreased absorbance and peak shift of amid I (C=O) stretches, slightly higher water uptake degree and significantly decreased in vitro degradation rate. Moreover, heat-treated ACG sponge maintained good 3-dimensional surface morphology and porous microstructure throughout 7 days, while non-heat-treated ACG sponge collapsed in less than 24 h. The human mesenchymal stromal cells (hMSCs) were shown to adhere and grow well on heat-treated ACG sponges. These results indicate that heat treatment is effective and safe to stabilize 3-dimensional ACG sponge biomaterial for tissue engineering.

Effects of changes in end-tidal carbon dioxide tension on oral tissue blood flow and tissue oxygen tension during remifentanil infusion in rabbits.

PURPOSE: The aim of this study was to investigate the effects of end-tidal carbon dioxide tension (ETCO2) changes during remifentanil infusion on mandibular bone marrow tissue blood flow (BBF), masseter muscle tissue blood flow (MBF), mandibular bone marrow tissue oxygen tension (PbO2) and masseter muscle tissue oxygen tension (PmO2) in rabbits. METHODS: Ten male tracheotomized Japan White rabbits were anesthetized and ventilated with sevoflurane. ETCO2 was adjusted to 30 mmHg. After baseline measurement, CO2 was added to the inhaled air, and ETCO2 was increased to 40 and 60 mmHg. Heart rate (HR), systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial pressure (MAP), BBF, MBF, PbO2, and PmO2 were recorded with and without remifentanil infusion at 0.4 µg/kg/min. RESULTS: Two-way repeated measures analysis of variance showed no interaction between ETCO2 and remifentanil in all variables. Remifentanil infusion produced decreases in HR, SBP, MAP, BBF and MBF compared with those without remifentanil infusion, while it did not affect DBP, PbO2 and PmO2. Elevation of ETCO2 from 30 to 60 mmHg produced decreases in HR and MBF, and increases in SBP, DBP, MAP and BBF, while it did not affect PbO2 and PmO2. CONCLUSION: PbO2 and PmO2 remained unchanged despite changes in BBF and MBF during ETCO2 change with or without remifentanil infusion.

Effects of Local Anesthetics With Vasoconstrictors on Dental Pulp Blood Flow and Oxygen Tension.

OBJECTIVE: The aim of this study was to investigate the changes in pulpal blood flow (PBF) and pulpal oxygen tension (PpulpO2) after injecting local anesthetics with vasoconstrictors. METHODS: Under general anesthesia, male Japanese White rabbits were injected with 0.6 mL of 2% lidocaine with 1:80,000 epinephrine (LE) or 3% propitocaine (prilocaine) with 0.03 IU felypressin (PF) at the apical area of the lower incisor. RESULTS: Relative to baseline, PBF and PpulpO2 significantly decreased 5 minutes after LE or PF injection as compared with saline. The decrease in PBF was significantly lower in the LE group than in the PF group. Although the LE group had a larger decrease in PpulpO2 relative to baseline than the PF group did, that difference was not significant. PBF and PpulpO2 recovered to baseline faster in the PF group than in the LE group. CONCLUSION: The injection of local anesthetic solutions containing vasoconstrictors (LE or PF) transiently caused significant decreases in PBF that resulted in significant decreases in PpulpO2. The recovery of PpulpO2 was faster than PBF regardless of the vasoconstrictor used.

Prenatal enzyme replacement therapy for Akp2 -/- mice with lethal hypophosphatasia.

Hypophosphatasia (HPP) is a congenital skeletal disease. Impairment of bone mineralization and seizures are due to a deficiency of tissue-nonspecific alkaline phosphatase (TNAP). Enzyme replacement therapy (ERT) is available as a highly successful treatment for pediatric-onset HPP. However, the potential for prenatal ERT has not been fully investigated to date. In this study, we assessed outcomes and maternal safety using a combinational approach with prenatal and postnatal administration of recombinant TNAP in Akp2 -/- mice as a model of infantile HPP. For the prenatal ERT, we administered subcutaneous injections of recombinant TNAP to pregnant mice from embryonic day 11.5-14.5 until delivery, and then sequentially to Akp2 -/- pups from birth to day 18. For the postnatal ERT, we injected Akp2 -/- pups from birth until day 18. Prenatal ERT did not cause any ectopic mineralization in heterozygous maternal mice. Both prenatal and postnatal ERT preserved growth, survival rate and improved bone calcification in Akp2 -/- mice. However, the effects of additional prenatal treatment to newborn mice appeared to be minimal, and the difference between prenatal and postnatal ERT was subtle. Further improvement of the prenatal ERT schedule and long-term observation will be required. The present paper sets a standard for such future studies.

Pentapartite fractionation of particles in oral fluids by differential centrifugation.

Oral fluids (OFs) contain small extracellular vesicles (sEVs or exosomes) that carry disease-associated diagnostic molecules. However, cells generate extracellular vesicles (EVs) other than sEVs, so the EV population is quite heterogeneous. Furthermore, molecules not packaged in EVs can also serve as diagnostic markers. For these reasons, developing a complete picture of particulate matter in the oral cavity is important before focusing on specific subtypes of EVs. Here, we used differential centrifugation to fractionate human OFs from healthy volunteers and patients with oral squamous cell carcinoma into 5 fractions, and we characterized the particles, nucleic acids, and proteins in each fraction. Canonical exosome markers, including CD63, CD9, CD133, and HSP70, were found in all fractions, whereas CD81 and AQP5 were enriched in the 160K fraction, with non-negligible amounts in the 2K fraction. The 2K fraction also contained its characteristic markers that included short derivatives of EGFR and E-cadherin, as well as an autophagosome marker, LC3, and large multi-layered vesicles were observed by electronic microscopy. Most of the DNA and RNA was recovered from the 0.3K and 2K fractions, with some in the 160K fraction. These results can provide guideline information for development of purpose-designed OF-based diagnostic systems.

Remifentanil infusion during desflurane anesthesia reduces tissue blood flow while maintaining blood pressure and tissue oxygen tension in the masseter muscle and mandibular bone marrow.

The aim of this study was to compare changes in tissue blood flow and tissue oxygen tension in the masseter muscle and mandibular bone marrow induced by remifentanil under desflurane or sevoflurane anesthesia. Eleven male tracheotomized Japan White rabbits were anesthetized with desflurane or sevoflurane under mechanical ventilation. The order of the inhalation of desflurane or sevoflurane was randomized. Desflurane or sevoflurane was administered at 1.0 minimum alveolar concentration and remifentanil was infused at 0.4 µg/kg/min. Observed variables included heart rate (HR), blood pressure (BP), common carotid artery blood flow (CCBF), mandibular bone marrow tissue blood flow (BBF), masseter muscle tissue blood flow (MBF), mandibular bone marrow tissue oxygen tension (PbO2), and masseter muscle tissue oxygen tension (PmO2). Two way repeated measures ANOVA showed no interaction between volatile anesthetics and remifentanil infusion except for MBF. There were significant differences in HR, SBP, DBP, MAP and CCBF between desflurane and sevoflurane groups. There were also significant differences in HR, SBP, DBP, MAP, CCBF, BBF and PbO2 before, during and after remifentanil infusion. Desflurane reduced tissue blood flow in the masseter muscle and mandibular bone marrow while better maintained HR and BP than sevoflurane. Under remifentanil infusion, although both anesthetics reduced tissue blood flow, tissue oxygen tension was maintained in masseter muscle and mandibular bone marrow.

High-Level Expression of Alkaline Phosphatase by Adeno-Associated Virus Vector Ameliorates Pathological Bone Structure in a Hypophosphatasia Mouse Model.

Hypophosphatasia (HPP) is a systemic skeletal disease caused by mutations in the gene encoding tissue-nonspecific alkaline phosphatase (TNALP). We recently reported that survival of HPP model mice can be prolonged using an adeno-associated virus (AAV) vector expressing bone-targeted TNALP with deca-aspartate at the C terminus (TNALP-D10); however, abnormal bone structure and hypomineralization remained in the treated mice. Here, to develop a more effective and clinically applicable approach, we assessed whether transfection with TNALP-D10 expressing virus vector at a higher dose than previously used would ameliorate bone structure defects. We constructed a self-complementary AAV8 vector expressing TNALP driven by the chicken beta-actin (CBA) promoter (scAAV8-CB-TNALP-D10). The vector was injected into both quadriceps femoris muscles of newborn HPP mice at a dose of 4.5 × 1012 vector genome (v.g.)/body, resulting in 20 U/mL of serum ALP activity. The 4.5 × 1012 v.g./body-treated HPP mice grew normally and displayed improved bone structure at the knee joints in X-ray images. Micro-CT analysis showed normal trabecular bone structure and mineralization. The mechanical properties of the femur were also recovered. Histological analysis of the femurs demonstrated that ALP replacement levels were sufficient to promote normal, growth plate cartilage arrangement. These results suggest that AAV vector-mediated high-dose TNALP-D10 therapy is a promising option for improving the quality of life (QOL) of patients with the infantile form of HPP.

Tissue-nonspecific alkaline phosphatase promotes the osteogenic differentiation of osteoprogenitor cells.

Tissue-nonspecific alkaline phosphatase (TNAP) is expressed in the calcification sites of the skeletal tissue. It promotes hydroxyapatite crystal formation by degrading inorganic pyrophosphate (PPi) and increasing inorganic phosphate (Pi) concentration. However, abnormalities in Alpl-/- mouse-derived osteoblasts are poorly understood, and the involvement of TNAP in osteoblast differentiation remains unclear. Therefore, in this study, we aimed to investigate the precise role of TNAP in osteoblast differentiation. TNAP inhibition by levamisole, a reversible TNAP inhibitor, suppressed the expression of osteoblast differentiation marker genes in wild-type osteoblastic cells. Alpl overexpression increased the expression of master osteoblast transcription factor genes runt-related transcription factor 2 (Runx2) and Sp7 and the mature osteoblast and osteocyte marker genes, bone γ-carboxyglutamate protein 2 (Bglap2) and dentin matrix protein 1 (Dmp1), respectively in Alpl-deficient osteoblastic cells. TNAP regulated Runx2 expression, which in turn regulated the expression of all other osteoblast markers, except Dmp1. Dmp1 expression was independent of RUNX2 but was dependent on extracellular Pi concentration in Runx2-deficient osteogenic cells. These results suggest that TNAP functions as an osteogenic differentiation regulator either by regulating Runx2 expression or by controlling extracellular Pi concentration.

Method for diagnosing neoplastic lesions by quantitative fluorescence value.

Fluorescence visualization devices (FVs) are useful for detecting malignant lesions because of their simple and noninvasive application. However, their quantitative application has been challenging. This study aimed to quantitatively and statistically evaluate the change in fluorescence intensity (FI) during the progression from normal epithelium to squamous cell carcinoma using a reproducible animal tongue carcinogenesis model. To establish this model, rats were treated with 50 ppm 4-Nitroquinoline 1-oxide (4NQO) in their drinking water for 10, 15, and 20 weeks. After 4NQO administration, each rat tongue was evaluated by gross observation, histology, and FI measurements. Fluorescence images were captured by FV, and ImageJ was used to measure FI, which was analyzed quantitatively and statistically. The establishment of a reproducible tumor progression model was confirmed, showing precancerous lesions (low-grade dysplasia [LGD]), early cancers (high-grade dysplasia/carcinoma in situ [HGD/CIS]), and advanced cancers (Cancer). This carcinogenesis model was quantitatively evaluated by FI. The FI of LGD stage was 54.6, which was highest intensity of all groups. Subsequently, the HGD/CIS and Cancer stages showed decreased FI (HGD/CIS: 46.1, Cancer: 49.1) and manifested as dark spots. This result indicates that FI had more variation and a wider range with increasing tumor progression. We demonstrated that FI migration and an uneven distribution are consistent with tumor progression. Since each step of tumor progression occurs reproducibly in this animal model, statistical evaluation was possible. In addition, tumor progression can be monitored by this new FI analysis method in humans.

Decreases in Oral Tissue Blood Flow Induced by Remifentanil Are Not Accompanied by Deterioration of Oral Tissue Oxygen Tension in Rabbits.

PURPOSE: The purpose of this study was to investigate the effects of remifentanil infusion on tissue blood flow and tissue oxygen tension in the mandibular bone marrow and masseter muscle in rabbits. In addition, changes in tissue oxygen consumption in those tissues during remifentanil infusion were investigated. MATERIALS AND METHODS: Sixteen male tracheotomized Japanese White rabbits were anesthetized with sevoflurane under mechanical ventilation. Under oxygen and air inhalation, fraction of inspiratory oxygen was set at 0.4 and remifentanil was infused at a rate of 0.4 µg ∙ kg-1 ∙ min-1. Measurements were performed before remifentanil infusion, 20 minutes after the start of remifentanil infusion, and 20 and 60 minutes after the completion of remifentanil infusion (n = 8). The observed variables included heart rate (HR), systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial pressure (MAP), mandibular bone marrow tissue blood flow (BBF), masseter muscle tissue blood flow (MBF), mandibular bone marrow tissue oxygen tension (PbO2), and masseter muscle tissue oxygen tension (PmO2). Another 8 rabbits were observed for arterial pH, lactate, base excess (BE), and tissue oxygen consumption in the region from which the retromandibular vein received venous blood. Measurements were performed before remifentanil infusion and 20 minutes after the start of remifentanil infusion. RESULTS: HR, SBP, DBP, MAP, BBF, and MBF decreased during remifentanil infusion. PbO2 increased 20 minutes after remifentanil infusion and returned to almost the baseline value 60 minutes after remifentanil infusion. PmO2 did not change throughout the experiment. The difference between the arterial oxygen content of the femoral artery and the venous oxygen content of the retromandibular vein decreased during remifentanil infusion. Arterial pH, lactate, and BE did not change during remifentanil infusion. CONCLUSIONS: Remifentanil decreased BBF and MBF but did not decrease PbO2 and PmO2. It is suggested that tissue oxygen consumption decreased during remifentanil infusion.

Remifentanil decreases oral tissue blood flow while maintaining internal carotid artery blood flow during sevoflurane anesthesia in rabbits.

The aim of this study was to investigate the effect of remifentanil infusion on oral tissue blood flow including submandibular gland tissue blood flow (SBF) and internal carotid artery blood flow (ICBF) in rabbits during sevoflurane anesthesia. Twelve male Japan White rabbits were anesthetized with sevoflurane and remifentanil. Remifentanil was infused at 0.2 and 0.4 µg/kg/min. Measurements included circulatory variables, common and external carotid artery blood flow (CCBF, ECBF), ICBF, tongue mucosal blood flow (TMBF), masseter muscle tissue blood flow (MBF), mandibular bone marrow tissue blood flow (BBF), tongue muscle tissue blood flow (TBF) and SBF. Vascular resistances for each tissue, including the tongue mucosa, masseter muscle, mandibular bone marrow, tongue muscle and submandibular gland, were calculated by dividing the mean arterial pressure by the respective tissue blood flow. Remifentanil infusion decreased oral tissue blood flow and circulatory variables. CCBF, ECBF and ICBF did not change. The calculated vascular resistance in each oral tissue, except for the tongue mucosa, increased in an infusion-rate-dependent manner. These results showed that remifentanil infusion reduced TMBF, MBF, BBF, TBF and SBF in an infusion-rate-dependent manner without affecting ICBF under sevoflurane anesthesia.

Effect of Topical Anesthesia Using an Adhesive Patch and Anesthetic Solution.

We analyzed trigeminal somatosensory evoked potentials (TSEP) to the alveolar mucosa to investigate the efficacy of an amide local anesthetic, 2% lidocaine hydrochloride with 12.5 µg/mL epinephrine (Lido treatment) as a topical anesthetic. Eighteen consenting healthy adult volunteers were enrolled. A volume of 0.06 mL of Lido, 0.06 g of 20% benzocaine, or 0.06 mL of physiological saline (control) was instilled onto a hemostatic adhesive patch, which was then applied to the alveolar mucosa at the maxillary right canine for 5 minutes. An electrical stimulus approximately 5 times that of the sensory threshold was applied using a surface stimulation electrode. The trigeminal somatosensory evoked potential was recorded immediately, 5 minutes, and 10 minutes after removal of the patch. Positive P125 and P310 peaks and negative N100 and N340 peaks were observed as a result of the electrical stimulation. A significant decrease in the percentage change in amplitude of N100-P125 was observed in the Lido treatment immediately, 5 minutes, and 10 minutes after patch removal. In the Lido treatment, trigeminal somatosensory evoked potential amplitude at N100-P125 decreased significantly, suggesting that topical anesthesia produced by an amide local anesthetic may have a topical anesthetic effect as potent as that produced by an ester local anesthetic.

Influence of lyophilization factors and gelatin concentration on pore structures of atelocollagen/gelatin sponge biomaterial.

This study aimed to investigate influences of lyophilization factors and gelatin concentration on pore structures of ACG sponge. ACG sponges of different freezing temperatures (-30, -80 and -196oC), freezing times (1, 2 and 24 h), gelatin concentrations (0.6%AC+0.15%G, 0.6%AC+0.6%G and 0.6%AC+2.4%G), and with 500 µM fluvastatin were fabricated. Pore structures including porosity and pore size were analyzed by scanning electron microscopy and ImageJ. The cytotoxic effects of ACG sponges were evaluated in vitro. Freezing temperature did not affect porosity while high freezing temperature (-30oC) increased pore size. The high gelatin concentration group (0.6%AC+2.4%G) had decreased porosity and pore size. Freezing time and 500 µM fluvastatin did not affect pore structures. The cytotoxicity and cell proliferation assays revealed that ACG sponges had no cytotoxic effects on human mesenchymal stromal cell growth and proliferation. These results indicate that ACG sponge may be a good biomaterial scaffold for bone regeneration.

Inhibition of the oxygen sensor PHD2 in the liver improves survival in lactic acidosis by activating the Cori cycle.

Loss of prolyl hydroxylase 2 (PHD2) activates the hypoxia-inducible factor-dependent hypoxic response, including anaerobic glycolysis, which causes large amounts of lactate to be released from cells into the circulation. We found that Phd2-null mouse embryonic fibroblasts (MEFs) produced more lactate than wild-type MEFs, as expected, whereas systemic inactivation of PHD2 in mice did not cause hyperlacticacidemia. This unexpected observation led us to hypothesize that the hypoxic response activated in the liver enhances the Cori cycle, a lactate-glucose carbon recycling system between muscle and liver, and thereby decreases circulating lactate. Consistent with this hypothesis, blood lactate levels measured after a treadmill or lactate tolerance test were significantly lower in Phd2-liver-specific knockout (Phd2-LKO) mice than in control mice. An in vivo (13)C-labeled lactate incorporation assay revealed that the livers of Phd2-LKO mice produce significantly more glucose derived from (13)C-labeled lactate than control mice, suggesting that blockade of PHD2 in the liver ameliorates lactic acidosis by activating gluconeogenesis from lactate. Phd2-LKO mice were resistant to lactic acidosis induced by injection of a lethal dose of lactate, displaying a significant elongation of survival. Moreover, oral administration of a PHD inhibitor improved survival in an endotoxin shock mice model. These data suggest that PHD2 is a potentially novel drug target for the treatment of lactic acidosis, which is a serious and often fatal complication observed in some critically ill patients.

Tissue Blood Flow During Remifentanil Infusion With Carbon Dioxide Loading.

The aim of this study was to investigate the effect of changes in end-tidal carbon dioxide tension (ETCO2) during remifentanil (Remi) infusion on oral tissue blood flow in rabbits. Eight male tracheotomized Japan White rabbits were anesthetized with sevoflurane under mechanical ventilation. The infusion rate of Remi was 0.4 µg/kg/min. Carbon dioxide was added to the inspired gas to change the inspired CO2 tension to prevent changes in the ventilating condition. Observed variables were systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial pressure (MAP), heart rate (HR), common carotid artery blood flow (CCBF), tongue mucosal blood flow (TBF), mandibular bone marrow tissue blood flow (BBF), masseter muscle tissue blood flow (MBF), upper alveolar tissue blood flow (UBF), and lower alveolar tissue blood flow (LBF). The CCBF, TBF, BBF, UBF, and LBF values were increased, while MBF was decreased, under hypercapnia, and vice versa. The BBF, UBF, and LBF values were increased, while the MBF value was decreased, under hypercapnia during Remi infusion, and vice versa. The BBF, MBF, UBF, and LBF values, but not the CCBF and TBF values, changed along with ETCO2 changes during Remi infusion.

Cervical sympathetic block prolongs the latency and reduces the amplitude of trigeminal somatosensory evoked potentials on the contralateral side.

PURPOSE: The purpose of this study was to investigate the latency and amplitude of trigeminal somatosensory evoked potentials to clarify how nerve function on the contralateral side is affected after cervical sympathetic block (CSB). METHODS: Subjects comprised 16 volunteers. For CSB, the tip of a needle was contacted with the transverse process of the sixth cervical vertebra on the right side, and lidocaine was injected. Trigeminal somatosensory evoked potentials were recorded bilaterally from C5/C6 scalp positions. Pupil diameters were also measured. Electrical stimulations were applied to the left-side lower lip, and trigeminal somatosensory evoked potentials waveforms derived from both sides of the scalp were recorded. Then, electrical stimulations were applied to the right-side of the lower lip, and recording was again performed. Recordings were performed at 5, 15, and 30 minutes after CSB. RESULTS: On the CSB side, pupil diameter decreased at 5 and 15 minutes after CSB. Trigeminal somatosensory evoked potentials at contralateral stimulation showed a prolongation of the latency in both P20 and N25 components on bilateral recording sites 5 and 15 minutes after CSB. Trigeminal somatosensory evoked potentials' amplitude at contralateral stimulation was smaller than at ipsilateral stimulation 5 minutes after CSB. CONCLUSIONS: Cervical sympathetic block prolongs the latency and reduces the amplitude of trigeminal somatosensory evoked potentials on the contralateral side.

Concomitant administration of nitrous oxide and remifentanil reduces oral tissue blood flow without decreasing blood pressure during sevoflurane anesthesia in rabbits.

PURPOSE: To determine whether continuous administration of nitrous oxide and remifentanil­either alone or together­alters blood flow in oral tissues during sevoflurane anesthesia. METHODS: Eight male tracheotomized Japanese white rabbits were anesthetized with sevoflurane under mechanical ventilation. Heart rate (HR), systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial pressure (MAP), common carotid arterial blood flow (CCBF), tongue mucosal blood flow (TMBF), mandibular bone marrow blood flow (BBF), masseter muscle blood flow (MBF), upper alveolar tissue blood flow (UBF), and lower alveolar tissue blood flow (LBF) were recorded in the absence of all test agents and after administration of the test agents (50 % nitrous oxide, 0.4 µg/kg/min remifentanil, and their combination) for 20 min. RESULTS: Nitrous oxide increased SBP, DBP, MAP, CCBF, BBF, MBF, UBF, and LBF relative to baseline values but did not affect HR or TMBF. Remifentanil decreased all hemodynamic variables except DBP. Combined administration of nitrous oxide and remifentanil recovered SBP, DBP, MAP, and CCBF to baseline levels, but HR and oral tissue blood flow remained lower than control values. CONCLUSIONS: Our findings suggest that concomitant administration of nitrous oxide and remifentanil reduces blood flow in oral tissues without decreasing blood pressure during sevoflurane anesthesia in rabbits.