Dedifferentiated Fat (DFAT) cells: A cell source for oral and maxillofacial tissue engineering.

Tissue engineering is a promising method for the regeneration of oral and maxillofacial tissues. Proper selection of a cell source is important for the desired application. This review describes the discovery and usefulness of dedifferentiated fat (DFAT) cells as a cell source for tissue engineering. Dedifferentiated Fat cells are a highly homogeneous cell population (high purity), highly proliferative, and possess a multilineage potential for differentiation into various cell types under proper in vitro inducing conditions and in vivo. Moreover, DFAT cells have a higher differentiation capability of becoming osteoblasts, chondrocytes, and adipocytes than do bone marrow-derived mesenchymal stem cells and/or adipose tissue-derived stem cells. The usefulness of DFAT cells in vivo for periodontal tissue, bone, peripheral nerve, muscle, cartilage, and fat tissue regeneration was reported. Dedifferentiated Fat cells obtained from the human buccal fat pad (BFP) are a minimally invasive procedure with limited esthetic complications for patients. The BFP is a convenient and accessible anatomical site to harvest DFAT cells for dentists and oral surgeons, and thus is a promising cell source for oral and maxillofacial tissue engineering.

Simulation training for medical emergencies in the dental setting using an inexpensive software application.

INTRODUCTION: Every dental provider needs to be educated about medical emergencies to provide safe dental care. Simulation training is available with simulators such as advanced life support manikins and robot patients. However, the purchase and development costs of these simulators are high. We have developed a simulation training course on medical emergencies using an inexpensive software application. The purpose of this study was to evaluate the educational effectiveness of this course. MATERIALS AND METHODS: Fifty-one dental providers participated in this study from December 2014 to March 2015. Medical simulation software was used to simulate a patient's vital signs. We evaluated participants' ability to diagnose and treat vasovagal syncope or anaphylaxis with an evaluation sheet and conducted a questionnaire before and after the scenario-based simulation training. RESULTS: The median evaluation sheet score for vasovagal syncope increased significantly from 7/9 before to 9/9 after simulation training. The median score for anaphylaxis also increased significantly from 8/12 to 12/12 (P < .01). For the item "I can treat vasovagal syncope/anaphylaxis adequately," the percentage responding "Strongly agree" or "Agree" increased from 14% to 56% for vasovagal syncope and from 6% to 42% for anaphylaxis with simulation training. CONCLUSIONS: This simulation course improved participants' ability to diagnose and treat medical emergencies and improved their confidence. This course can be offered inexpensively using a software application.

Formation of bone-like apatite enhanced by hydrolysis of octacalcium phosphate crystals deposited in collagen matrix.

It has been shown that granules of synthetic octacalcium phosphate (OCP) or the composites with collagen are capable of enhancing bone regeneration, accompanied by a gradual conversion from OCP to apatite with time. The present study was designed to investigate whether formation of bone-like apatite can be accelerated by OCP deposited throughout collagen matrix (OCP collagen complex, OCC) immersed in simulated body fluid (SBF). The formation of bone-like apatite has been suggested to be essential to induce osteoconductivity of various substrates. The formation of OCP in collagen solution was investigated in calcium or phosphate ions in the range between 22.5 and 142.5 mM and pH 6.26 and 8.56. X-ray diffraction, Fourier transform infrared spectroscopy, and scanning electron microscopy (SEM) showed that condition to nucleate OCP was limited to that of a solution with Ca/P 0.43 around pH 7.16 in the presence of collagen. OCP was shown to be formed throughout the collagen matrix by SEM observation. The immersion of OCC in SBF up to 10 days enhanced apatite crystal deposition, probably through OCP-apatite conversion: the apatite formation in OCC took place within only 1 day. The present study indicated that the existence of OCP deposited throughout the collagen matrix promotes bone-like apatite formation under physiological condition.

Capnography Prevents Hypoxia during Sedation for Dental Treatment: A Randomized Controlled Trial.

Intravenous sedation is useful for dental treatment in patients with intellectual disabilities. However, it is often necessary to manage such patients with deep sedation because their cooperation cannot be obtained. During deep sedation, undetected hypoventilation can lead to severe complications, such as hypoxia. Recently, capnographic monitoring has been advocated as a useful technique for preventing hypoxia during sedation. This randomized control trial evaluated whether the use of capnography reduces the incidence of hypoxia during the deep sedation of patients for dental treatment. This study involved patients with intellectual disabilities who underwent dental treatment under sedation. The subjects were randomized to the intervention group (I-group) or control group (C-group). All of the patients underwent routine monitoring, as well as bispectral index (BIS) and capnographic monitoring; however, only an independent observer had access to the patients' capnographic data during the dental procedures. Sedation was maintained at a BIS of 50 to 70 by administration of propofol. In the I-group, the independent observer signaled to the dental anesthesiologist if the capnogram indicated that the patient had been suffering from alveolar hypoventilation or apnea for >15 s. In the C-group, the observer signaled to the dental anesthesiologist if the capnogram indicated that the patient had been suffering from alveolar hypoventilation or apnea for >60 s. In both groups, the dental anesthesiologists responded to the signals using appropriate airway management strategies. The primary endpoint of this study was the incidence of hypoxia during dental treatment, which was defined as oxygen saturation of <95%. Hypoxemic episodes occurred in 13.4% and 34.8% of cases in the I-group and C-group, respectively. The incidence of hypoxia was significantly lower in the I-group. These results suggest that capnographic monitoring during deep sedation for dental treatment prevents hypoxemic episodes by allowing the early detection of hypoventilation. Knowledge Transfer Statement: This is the first randomized controlled trial to examine whether the use of capnography reduces the incidence of hypoxia during deep sedation for dental treatment. The findings of this study can be used by clinicians to aid decision-making regarding dental sedation standards at individual clinics. Moreover, they can be used as high-level evidence during the production or updating of clinical guidelines for dental sedation by leading associations.

Novel drug-delivery stent: intravascular ultrasound observations from the first human experience with the QP2-eluting polymer stent system.

BACKGROUND: The aim of this study was to use serial intravascular ultrasound (IVUS) to evaluate the long-term effect of stent-based 7-hexanoyltaxol (QP2, a taxane analogue) delivery on neointimal tissue growth within the stent and on vessel dimensions at the adjacent reference segments. METHODS AND RESULTS: Serial IVUS analyses (immediately after intervention and at follow-up at 8.3 months) were performed in 15 native coronary lesions treated with the QuaDS-QP2 stent. IVUS measurements were performed at 8 cross-sections in each target segment (4 cross-sections within the stent and 2 cross-sections in each reference segment). At baseline, no significant plaque protrusion or thrombus was detected in the target segment. Mild incomplete stent apposition and edge dissection were observed in one and two cases, respectively. Percent expansion of the stent (minimum stent area/average reference lumen area) was 96.0+/-21.7%. At follow-up, mean neointimal area within the stent was 1.2+/-1.3 mm(2), and mean cross-sectional narrowing (neointimal area/stent area) was 13.6+/-14.9%. At the vessel segments immediately adjacent to the stent, a significant increase in plaque area (1.9+/-2.6 mm(2), P=0.001) was observed, but vessel area remained unchanged. However, no patients showed clinically significant in-stent or edge restenosis (diameter stenosis >/=50%) during the follow-up period. CONCLUSIONS: The first human experience with the new drug-delivery stent showed a minimal amount of neointimal proliferation in the stented segment. Late lumen loss at the reference sites adjacent to the stent was acceptable and predominantly due to plaque proliferation.

Chorioretinal temperature monitoring during transpupillary thermotherapy for choroidal neovascularisation.

AIMS: To investigate the difference in temperature rise between normal choroid and choroidal revascularisation (CNV) during transpupillary thermotherapy (TTT) and the relation between laser spot size and power in the rat fundus. METHODS: A modified slit lamp, which was installed with two laser wavelengths (490 nm for illumination and fluorescein excitation and 810 nm for hyperthermia), was developed for TTT and temperature monitoring. Temperature rise during TTT was monitored by observing fluorescence released from thermosensitive liposomes encapsulating carboxyfluorescein. Two types of liposomes were prepared; their phase transition temperatures were 40 degrees C and 46 degrees C, respectively. Laser power settings required to observe fluorescence released from 46 degrees C liposome in normal choroid or CNV were compared. Next, the power settings with 0.5 mm and 0.25 mm spot sizes were compared following administration of 40 degrees C liposome or 46 degrees C liposome. RESULTS: The minimum power values when release from 46 degrees C liposome was observed showed a significant difference in distribution of power values between normal choroid and CNV. CNV required significantly higher power than normal choroid. With 40 degrees C liposome, the power was 9.7 (1.9) mW (mean (SD)) at a spot size of 0.25 mm, and 12.1 (1.6) mW at 0.5 mm, respectively. When using 46 degrees C liposome, the power setting was 10.2 (1.2) mW at a spot size of 0.25 mm, and 14.6 (2.2) mW at 0.5 mm, respectively. CONCLUSIONS: CNV demonstrated varying heat conduction, compared with normal choroid. Laser power required to raise the temperature should not necessarily be doubled, even when the spot size is doubled. Close attention should be given to the selection of power settings when performing TTT for CNV.

Two patients treated with simeprevir plus pegylated-interferon and ribavirin triple therapy for recurrent hepatitis C after living donor liver transplantation: case report.

We previously reported our data on telaprevir (TVR) used in combination with pegylated-interferon and ribavirin (PEG-IFN/RBV) for the treatment of recurrent hepatitis C virus (HCV) genotype 1 infection after liver transplantation (LT). TVR substantially increases the blood levels of immunosuppressive agents such as cyclosporine and tacrolimus for drug-drug interactions. On the other hand, the effect of simeprevir (SMV) on the blood levels of these immunosuppressive agents is unclear. We report 2 patients who achieved viral responses with little effect on the blood levels of cyclosporine and tacrolimus using SMV plus PEG-IFN/RBV treatment. The first was a 71-year-old woman with HCV-related liver cirrhosis and hepatocellular carcinoma who failed to respond to PEG-IFN/RBV after living donor LT. She was treated with 40 mg/d of cyclosporine, and received SMV plus PEG-IFN/RBV treatment. The second was a 65-year-old man with HCV-related liver cirrhosis who failed to respond to PEG-IFN/RBV after living donor LT. He was treated with 3 mg/d of tacrolimus, and received SMV plus PEG-IFN/RBV treatment. Serum HCV RNA became undetectable using TaqMan polymerase chain reaction (PCR) test after 4 weeks of treatment in both patients, and no remarkable fluctuation in blood concentration was observed either in cyclosporine or tacrolimus during the 12 weeks of SMV treatment. Completion of 12-week SMV triple therapy was followed by PEG-IFNα2b plus RBV, and both patients achieved sustained virological response 12 weeks after the end of treatment. SMV plus PEG-IFNRBV treatment showed a remarkable viral response with little effect on blood levels of immunosuppressive agents for recurrent HCV genotype 1 infection after LT.

In vivo gene transfer into the retina mediated by a novel liposome system.

PURPOSE: To determine whether a reporter gene can be introduced into rat and mouse retinas in vivo using the authors' novel liposome system. METHODS: Cytomegalovirus-promoted LacZ genes and nonhistone nuclear protein, high mobility group 1 (HMG1), were coencapsulated in liposomes by agitation and sonication. The liposomes were then coated with the envelope of inactivated hemagglutinating virus of Japan (HVJ; Sendai virus) by fusion (HVJ liposome). The HVJ liposome solution was injected into the vitreous cavity of adult Sprague-Dawley rats (n = 30) or the subretinal space of adult CD-1 mice (n = 42). LacZ expression was assessed by beta-galactosidase assay. RESULTS: LacZ expression was demonstrated in the photoreceptors as long as 30 days after intravitreal and subretinal injections. beta-Gal activity was observed also in neurons and glial cells in the ganglion cell layer of the intravitreally injected rats and, although at lower intensity, in the retinal pigment epithelium of both animals. No inflammation or toxic effects secondary to the HVJ liposomes were detected on histologic examination. CONCLUSIONS: In vivo transfer and expression of a reporter gene into adult mammalian retina can be achieved using HVJ liposomes. This method offers a promise as a nonviral system for in vivo gene transfer into adult mammalian neural retina.

Introduction of DNA into the rat and primate trabecular meshwork by fusogenic liposomes.

PURPOSE: To evaluate the feasibility of introducing exogenous genes and phosphorothioate oligonucleotides into the anterior chamber tissues of rats and monkeys using the authors' fusogenic liposomes. METHODS: Hemagglutinating virus of Japan liposomes containing LacZ DNA-high-mobility group 1 complexes or fluorescein isothiocyanate (FITC)-labeled phosphorothioate oligonucleotides were prepared and injected into the anterior chambers of rats (3 microliters) and rhesus monkeys (30 microliters). The expression of LacZ DNA was visualized histochemically by beta-Galactosidase assay and was followed for as long as 60 days in rats and 30 days in monkeys. FITC-labeled phosphorothioate oligonucleotides were observed by fluorescence microscopy for as long as 14 days in rats and 7 days in monkeys. RESULTS: Injection of LacZ DNA-high-mobility group 1 complexes encapsulated in hemagglutinating virus of Japan liposomes resulted in blue staining in the trabecular meshwork and iris-ciliary body of rats and selectively in the trabecular meshwork of monkeys at the concentrations used. This LacZ expression lasted for as long as 14 days after injection in both animals. Phosphorothioate oligonucleotides (3 microM) also were introduced into the rat trabecular meshwork and iris-ciliary body and into the primate trabecular meshwork when encapsulated in hemagglutinating virus of Japan liposomes, although the injection of naked FITC-labeled phosphorothioate oligonucleotides at the same concentration resulted in little fluorescence in any anterior chamber tissue. CONCLUSIONS: This study shows that the use of hemagglutinating virus of Japan liposomes can transfer LacZ DNA and phosphorothioate oligonucleotides to adult rat and primate trabecular meshwork. This system may enable progress in glaucoma research and in the development of nonviral somatic gene therapy of the trabecular meshwork to treat glaucoma.

Microspheres of biodegradable polymers as a drug-delivery system in the vitreous.

Microspheres of biodegradable polymers were evaluated as a potential controlled-release drug-delivery system in the vitreous. The microspheres were prepared with polymers of poly(lactic acid) or copolymers of glycolic acid and lactic acid. The release of 5-fluorouracil (5-FU) from the microspheres was studied in vitro. Poly(lactic acid) microspheres released 70-85% of total 5-FU over 7 days. Microspheres of polymers with a smaller molecular weight released the drug more rapidly. Copolymer microspheres released 98% of 5-FU over 2 days. The rate of drug release was controllable by changing the molecular weight of the polymers or using a matrix of copolymer. The intravitreal kinetics of the microspheres were studied in ten rabbits in vivo. A suspension of microspheres was injected into the vitreous cavity of five normal eyes and five vitrectomized eyes. By 48 +/- 5.2 days after injection, the microspheres disappeared from the vitreous cavity in the five normal eyes. Clearance from the vitreous cavity was accelerated in the five rabbits that underwent vitrectomy (14 +/- 2.4 days; P less than 0.001). No difference was found in the b waves of electroretinograms before and after injection of the microspheres. The histologic study showed no abnormal findings as a result of the injection. These results suggested that microspheres of biodegradable polymers may be a potential delivery system for the controlled release of drugs in the vitreous.

Targeted delivery of anti-angiogenic agent TNP-470 using water-soluble polymer in the treatment of choroidal neovascularization.

PURPOSE: The conjugation of drugs with water-soluble polymers such as poly(vinyl alcohol) (PVA) tends to prolong the half-life of drugs and facilitate the accumulation of drugs in tissues involving neovascularization. The purpose of this study was to evaluate the effect of TNP-470-PVA conjugate on the proliferation of endothelial cells in vitro and on experimental choroidal neovascularization (CNV) in vivo. METHODS: TNP-470 was conjugated in PVA by a dimethylaminopyridine-catalyzed reaction. The effects of TNP-470-PVA and free TNP-470 on the proliferation of human umbilical vein endothelial cells (HUVECs) and bovine retinal pigment epithelial cells (BRPECs) were evaluated by the tetrazolium-based colorimetric assay (XTT assay). Experimental CNV was induced by subretinal injection of gelatin microspheres containing basic fibroblast growth factor, into rabbits. Thirty rabbits were intravenously treated either with TNP-470-PVA (n = 8), free TNP470 (n = 5), free PVA (n = 5), or saline (n = 12) daily for 3 days, 2 weeks after implantation of gelatin microspheres. Fluorescein angiography was performed to detect the area with CNV, and the evaluation was made by computerized measurement of digital images. These eyes were also examined histologically. To observe the accumulation of conjugate, 3 rabbits with CNV received rhodamine B isothiocyanate-binding PVA (RITC-PVA), and the lesion was studied 24 hours later by fluorescein microscopy. RESULTS: The TNP-470-PVA inhibited the growth of HUVECs, similar to that of free TNP-470. The BRPECs were less sensitive to TNP-470-PVA than were the HUVECs. TNP-470-PVA significantly inhibited the progression of CNV in rabbits (P = 0.001). Histologic studies at 4 weeks after treatment demonstrated that the degree of vascular formation and the number of vascular endothelial cells in the subretinal membrane of the eyes treated with TNP-470-PVA were less than those of the control eyes. RITC-PVA remained in the area with CNV 24 hours after administration. CONCLUSIONS: These results suggest that TNP-470-PVA inhibited the proliferation of HUVECs more sensitively than that of BRPECs, and the targeted delivery of TNP-470-PVA may have potential as a treatment modality for CNV.

A new vitreal drug delivery system using an implantable biodegradable polymeric device.

PURPOSE: The authors evaluated the feasibility of using an implantable biodegradable polymeric device to deliver drugs into the vitreous humor. METHODS: Two types of devices were prepared by compression-molding polymers of poly(DL-lactic acid) of two different molecular weights. The molecular weights of the poly(DL-lactic acid) used were 5,600 (device-1) and 9,100 (device-2). Sodium fluorescein (NaF) served as a hydrophilic drug marker. The release of the dye from the devices was studied in vitro. The intravitreal kinetics of NaF was evaluated in rabbits in vivo by fluorophotometry. The eyes were evaluated electrophysiologically and histologically to determine if there were toxic effects. RESULTS: Device-1 and device-2 released NaF for more than 25 and 45 days, respectively, in vitro. Detectable concentrations of NaF were present in the vitreous up to 17 days (device-1) and 28 days (device-2). Both types of devices were well tolerated, with no noted toxic effects. CONCLUSIONS: These results suggested that this device may be a potentially effective system to deliver drugs in the vitreous.

The effect of hypoxic depression on genioglossal muscle activity in healthy subjects and obstructive sleep apnea patients.

We investigated whether selective depression of the genioglossus muscle (GG) activity could be associated with hypoxic ventilatory depression, which is developed in the late phase of the biphasic ventilatory response during sustained hypoxia. Eleven control subjects and 10 patients with obstructive sleep apnea syndrome (OSAS) were examined by an isocapnic sustained hypoxia test for 20 minutes. Ventilatory parameters and electromyographic (EMG) activities were recorded from GG (EMGGG) and diaphragm (EMGDIA). Hypoxic depression in minute ventilation (V1) and EMGDIA occurred when compared between the peak value and the value at late period in both groups. On the contrary, depression in EMGGG was not observed in controls (86% of the peak value), whereas EMGGG was strikingly depressed in OSAS (39% of the peak value). We conclude that sustained hypoxia reduces the activity of GG in OSAS, but not in control subjects. These data suggest that the lack of a compensatory response of GG to sustained hypoxia is possibly responsible for the pathogenesis of OSA.

In vivo delivery of phosphorothioate oligonucleotides into murine retina.

OBJECTIVES: To determine the fate of phosphorothioate oligonucleotides (S-ODNs), which are commonly used for antisense strategy, in murine retina in vivo with the use of fluorescein isothiocyanate (FITC)-labeled S-ODNs, and to evaluate our fusogenic liposome system that may facilitate the delivery of S-ODNs. METHODS: The FITC-labeled S-ODNs were encapsulated in liposomes, which were then coated with the envelope of inactivated hemagglutinating virus of Japan (HVJ; Sendai virus) by fusion (HVJ liposomes). Intravitreal injection of naked FITC-labeled S-ODNs or of the HVJ liposomes was done in ICR mice. After fixation, cryosections and flat-mounted retinas were prepared and examined by fluorescence microscopy. RESULTS: Injection of naked FITC-labeled S-ODNs at 3 micromol/L exhibited weak fluorescence in 13% of the cells in the ganglion cell layer. When the concentration was increased to 30 micromol/L, high fluorescence was seen in 59% of cells in the ganglion cell layer at this time. This fluorescence diminished within a day. In contrast, injection of HVJ liposomes containing FITC-labeled S-ODNs at 3 micromol/L resulted in high fluorescence in 44% of the cells in the ganglion cell layer at 1 hour, and this fluorescence lasted for up to 3 days. This treatment also resulted in high fluorescence within retinal vessel walls, and weak fluorescence in photoreceptor cells. CONCLUSIONS: Intravitreally injected S-ODNs were rapidly eliminated from neural retina, and the use of HVJ liposomes could improve the delivery of S-ODNs. This method may be a potentially useful system for the antisense-based therapies for retinal diseases.

Scleral plug of biodegradable polymers for controlled drug release in the vitreous.

We designed a new device, a scleral plug, that releases drugs into the vitreous after being implanted and fixed at the pars plana. Use of the plug for provision of doxorubicin hydrochloride was evaluated in rabbits. The scleral plug (8.5 mg) was made of poly(lactic-glycolic acid) (molecular weight, 40,000 daltons) containing 1% doxorubicin. Vitreous concentrations of doxorubicin were measured after the implantation. In vitro studies showed that the plug released 26% of the drug during 4 weeks. In vivo studies demonstrated that the concentration in the vitreous humor was maintained at a therapeutic range for longer than 4 weeks. No substantial toxic reactions were observed by electroretinographic and histopathologic evaluations. Our findings suggested that a scleral plug made of biodegradable polymers is a promising device for a controlled drug-release system in the vitreous.

An assessment of nasal functions in control of breathing.

Breathing pattern and steady-state CO2 ventilatory response during mouth breathing were compared with those during nose breathing in nine healthy adults. In addition, the effect of warming and humidification of the inspired air on the ventilatory response was observed during breathing through a mouthpiece. We found the following. 1) Dead space and airway resistance were significantly greater during nose than during mouth breathing. 2) The slope of CO2 ventilatory responses did not differ appreciably during the two types of breathing, but CO2 occlusion pressure response was significantly enhanced during nose breathing. 3) Inhalation of warm and humid air through a mouthpiece significantly depressed CO2 ventilation and occlusion pressure responses. These results fit our observation that end-tidal PCO2 was significantly higher during nose than during mouth breathing. It is suggested that a loss of nasal functions, such as during nasal obstruction, may result in lowering of CO2, fostering apneic spells during sleep.

Degradation of benzyl ether bonds of lignin by ruminal microbes.

We examined microbial activity in the rumen to cleave benzyl ether bonds of lignin model compounds that fluoresced when the bonds were cleaved. 4-Methylumbelliferone veratryl ether dimer was degraded completely within 8 h even in the presence of fungicidal antibiotics, but no significant degradation occurred with bactericidal antibiotics. Degradation of a phenolic beta-O-4 trimer incorporating 4-methylumbelliferone by a benzyl ether linkage was stimulated by ruminal microbes, although its corresponding non-phenolic model compound, 1-(4-ethoxy-3-methoxyphenyl)-1-O-(4-methylumbelliferyl)-2-(2-methoxyp henoxy)-3-propanol, was not degraded. A coniferyl dehydrogenation polymer bearing fluorescent beta-O-4 benzyl ether that contains both phenolic and non-phenolic benzyl ether bonds was partially degraded (about 20%) in 48 h. These results suggest that ruminal microbes decompose benzyl ether linkages of lignin polymers under anaerobic conditions.

Extracellular lipid peroxidation of selective white-rot fungus, Ceriporiopsis subvermispora.

Ceriporiopsis subvermispora is capable of decomposing lignin without penetration of enzymes into wood cell walls. To elucidate the mechanism of lignolysis at a site far from enzymes, peroxidation of low molecular mass compounds produced by this fungus was analyzed. C. subvermispora produced free 9,12-octadecadienoic, 9-octadecenoic, 11-octadecenoic, hexadecanoic and octadecanoic acids, predominantly at an early stage of cultivation on wood meal cultures. In prolonged cultivation period after 2 weeks, the amount of intact fatty acids decreased with increasing organic hydroperoxide and TBARS production. These results suggest that lignin degradation by C. subvermispora is related to extracellular lipid peroxidation.

Rat circadian rhythms entrain to a descending saw-tooth light intensity cycle.

We reexamined whether rat circadian rhythms entrained to the light intensity cycle of a descending saw-tooth (ST-d) form, in which illuminance decreased rectilinearly from 300 lx to 0 lx in 24 h, and abruptly returned to 300 lx (lights-on time). Ambulation, drinking and subcutaneous body temperature were simultaneously monitored in 5 intact, 5 pinealectomized and 5 orchiectomized rats. Additionally, sleep was monitored in the intact rats. In all the rats, entrainment was confirmed during 65 days' exposure to the ST-d cycle. The waveforms of the entrained rhythms were much modified compared with those during LD 12:12. The estimated activity periods of the entrained rhythms straddled the lights-on time of the ST-d cycle. In all the groups of rats, administration of a single ST-d cycle in constant dim red light produced only delay shifts irrespective of its circadian phase, and there was no significant circadian variation in the magnitude of phase shifts. The results indicate that rat circadian rhythms entrain to the ST-d cycle with an unexpected phase position, which cannot be explained by the phase-response curve.

Circadian behavioral rhythms during various light-intensity cycles in rats.

Sleep, ambulation and drinking were recorded on 5 kinds of light-intensity cycles fluctuating between 300 lux and 0 lux with a 24-hr period in rats. The waveforms of the cycles were rectangular wave (RA), sinusoidal wave (SO), triangular wave (TA), descending saw-tooth wave (ST-d) and ascending saw-tooth wave (ST-a). Each condition was maintained for 28 days. Thereafter, the rats were released into constant darkness (DD) for 20 days. The circadian behavioral rhythms clearly entrained to RA, SO, TA and ST-a, but not to ST-d. The waveforms of the behavioral rhythms varied depending on those of the fluctuating light-intensity cycles, and were quite different from those in RA and DD. Daily amounts of slow wave sleep and paradoxical sleep were hardly affected by the lighting conditions, while those of ambulation and drinking were decreased on the other light-intensity cycles than RA. These results present new aspects of entrainment and masking of circadian rhythm by light.