Macrophage populations show an M1-to-M2 transition in an experimental model of coronal pulp tissue engineering with mesenchymal stem cells.

AIM: To assess M1/M2 macrophage phenotypes in a coronal pulp regeneration model in rats, under the hypothesis that there are dynamic M1/M2 phenotype changes during the different stages of the pulp regeneration. METHODOLOGY: The maxillary first molars of Wistar rats were pulpotomized, and biodegradable hydrogel-made scaffolds carrying rat bone marrow mesenchymal stem cells were implanted in the pulp chamber. After 3, 7 and 14 days, samples were processed for (i) histological analysis and double immunoperoxidase staining for CD68 (a general macrophage marker) and one of either CCR7 (an M1 marker), CD163 (an M2 marker) or CD206 (an M2 marker); (ii) real-time PCR for AIF1 (an M1 marker), CD163, CD206, IL-10 and TNF-α mRNA expression; and (iii) Western blotting for the detection of CD68, CCR7 and CD206 proteins. RESULTS: Histological analysis of the implanted region revealed sparse cellular distribution at 3 days, pulp-like tissue with a thin dentine bridge-like structure at 7 days, and dentine bridge-like mineralized tissue formation and resorption of most scaffolds at 14 days. CCR7+ macrophages had the highest density at 3 days, and then significantly decreased until 14 days (P < 0.05). In contrast, M2 marker (CD163 or CD206) expressing macrophages had the lowest density at 3 days and significantly increased until 14 days (P < 0.05). AIF1 and TNF-α mRNA levels, and CD68 and CCR7 protein levels were highest at 3 days. CD163 and CD206 mRNA levels, and CD206 protein levels increased with time and showed the highest at 14 days. IL-10 mRNA was highest at 3 days, decreased at 7 days and increased at 14 days. CONCLUSIONS: Macrophages in the regenerating pulp tissue underwent a distinct transition from M1-dominant to M2-dominant, suggesting that the M1-to-M2 transition of macrophages plays an important role in creating a favourable microenvironment necessary for pulp tissue regeneration.

Establishment of a tetraploid Meth-A cell line through polyploidization by demecolcine but not by staurosporine, K-252A and paclitaxel.

Polyploid cells are made by DNA reduplication without cell division, however, it is not easy to establish polyploid mammalian cell lines. It is worth studying the difference in cell character between hyperploid and parent cell lines. Meth-A cells were polyploidized by demecolcine, K-252a, staurosporine and paclitaxel. The cell-cycle responses of highly polyploid Meth-A cells after the removal of the drugs were examined by flow cytometry (FCM). Meth-A cells were highly polyploidized by these drugs. The polyploid Meth-A cells gradually decreased in ploidy after the drug release. A tetraploid Meth-A cell line was established only from the demecolcine-induced polyploid Meth-A cells. The duration of G1, S and G2/M phases of the tetraploid cell line were mostly the same as those of the parent diploid cells, except that the G2/M phase was 1.5 h longer. The chromosome number of tetraploid Meth-A cell line was about twice of the diploidy. A tetraploid Meth-A cell line was established.

Spectral analysis of circadian rhythms in heart rate variability of dogs.

OBJECTIVE: To determine characteristics of power spectral analysis of heart rate variability (HRV) during a 24-hour period in dogs and to evaluate the effects of vagal and sympathetic tone on HRV ANIMALS: 16 healthy adult Beagles. PROCEDURE: Power spectral analysis of HRV was conducted, using 24-hour ambulatory ECG recordings. Circadian rhythms were evaluated in terms of absolute units of low-frequency (LF) and high-frequency (HF) powers, their ratio (LF:HF), and their adjusted (normalized) units (LF[norm] and HF[norm]). Three or 4 dogs were used for simultaneous measurement of heart rate and respiratory waveform as well as to evaluate treatment (propranolol, atropine, or both) administered to cause blockade of the autonomic nervous system. RESULTS: Values for LF and HF powers, LF:HF, LF(norm), and HF(norm) had obvious rhythmicity in clinically normal dogs. The HF power of HRV in dogs was extremely high, compared with that of other species, and HF peaks corresponded to peaks obtained from respiratory waveforms. Blockade of the autonomic nervous system documented that HRV in dogs was mostly attributable to vagal activity. CONCLUSION AND CLINICAL RELEVANCE: We determined characteristics of power spectral analysis of HRV in dogs, including circadian rhythm of the autonomic nervous system. Power spectral analysis of HRV may provide a useful noninvasive technique for assessing the effect of drugs on activity of the autonomic nervous system in dogs.

D0870, an antifungal agent, induces reverse use-dependent QT prolongation in dogs.

We previously reported that D0870 induced QT prolongation and sudden death due to torsades de pointes (TdP) in dogs and that catecholamines played an important part in the development of the sudden death. In the present study, we analyzed in detail the ambulatory electrocardiographic recordings obtained from the just-mentioned study to elucidate the mechanism of the onset of TdPs and conducted an in vitro study using isolated canine Purkinje fibers to assess the effect of D0870 on repolarization. The hearts with TdPs observed before the sudden death showed a higher sinus rate for 5 and 10 sec before the onset, a shorter coupling interval, and a higher ventricular tachycardia rate compared with those having the non-sustained TdPs. These findings suggest that D0870-induced fatal TdPs may be provoked by a triggered activity developed from delayed after depolarizations. In contrast, as the pause-dependent, non-sustained TdPs in bradycardia showed a typical "short-long-short" sequence, they may be developed from early afterdepolarization . Moreover, the results of the in vitro study supported our contention that D0870 induced QT prolongation in a reverse use-dependent manner in vivo and suggested that it may inhibit not only rapidly activating delayed rectifier potassium current (Ik(r)) but also L-type Ca current (I(ca-L)).

Influences of catecholamines on the sudden death induced in dogs by an antifungal agent, D0870.

We previously reported the occurrence of QT prolongation and sudden death owing to torsades de pointes (TdP) in dogs treated with D0870, an antifungal agent. In the present study, we evaluated the influences of epinephrine and isoproterenol on the onset of TdP each time D0870 was given to 6 anesthetized open-chest dogs at a dosage of 20 mg/kg, 5 times every 40 minutes, by the simultaneous measurements of surface electrocardiogram and epicardial monophasic action potential (MAP). D0870 alone induced noticeable prolongation of the QT interval and action potential duration (APD), but neither ventricular premature contraction (VPC) nor sudden death. In contrast, the additional administration of the catecholamines induced a greater shortening of APD during the later phase of repolarization than during its earlier one and VPCs in all dogs tested, and sudden deaths owing to TdPs in 4 of the 6 dogs treated with D0870. These results suggest that D0870 alone does not induce TdP but that catecholamines play an important part in the development of sudden death induced by D0870 in dogs.

Location and severity of UVB irradiation damage in the rat lens.

We investigated the location and severity of lens opacities and epithelial alterations following ultraviolet-B (UVB) irradiation in vivo, using Brown Norway rats. A group of 9 rats received 65 mJ/cm2 UVB irradiation from overhead lamps every 6 days. Lens changes were documented and evaluated by an anterior eye segment analysis system. Lens epithelial cells were examined postmortem in flat preparations. After 8 weeks of the irradiation schedule (total dose: 0.6 J/cm2), an anterior polar opacity was apparent; at 16 weeks, the opacities had progressed more deeply into the cortex. At postmortem examination, cells in the central region displayed disorganization, clumping, some pyknotic nuclei and mitosis. There were deeper opacities and cell damage was more severe above the central horizontal plane than below it. This present study demonstrated that UVB damage differed in the superior and inferior parts divided by a horizontal plane through the lens anterior pole, when the UVB source was above and there was no reflection from below or laterally. The lens epithelial cells, and associated lens fibers, are the first target of UVB irradiation.

QT corrected for heart rate and relation between QT and RR intervals in beagle dogs.

The beagle dog has been widely used in cardiovascular research, but the adequacy of QT prediction formulas in dogs over a wide range of RR intervals has not been evaluated sufficiently. We investigated the QT-RR relation in beagles by analysis of the QT and preceding RR intervals obtained from 24-h ambulatory electrocardiograms. The acceptability of 14 QT prediction formulas was evaluated by use of 100-150 selected pairs of QT-RR points per animal in seven male and seven female beagles. The accuracy of fit with the measured data was assessed according to the minimum Akaike information criterion. The best fit was given by the logarithmic and inverse Kovács' formulas among one- and two-parameter linear regression equations, respectively. Exponential formulas produced a better fit than did the linear regression formulas, but are impractical because of the complicated interpretation of parameters due to the nonlinearity. In addition, the results obtained under physiological conditions were also confirmed by those of the pharmacological intervention study with disopyramide. Consequently, we propose a one-parameter logarithmic formula (QTc= log600 x QT/logRR) for correcting the QT interval for a heart rate of 100 bpm and the inverse Kovács' formula for evaluating a reverse-use-dependency of QT prolongation.

Inhibition of steroid-induced cataract in rat eyes by administration of vitamin-E ophthalmic solution.

The efficacy of a vitamin-E (VE) ophthalmic solution was evaluated on a newly developed rat steroid-induced cataract model. Brown Norway rats irradiated with 2 Gy X-ray, right eyes only, were divided into 5 groups: the control group; 2 steroid (1 mg/kg/day)-treated groups with topic (Top) and systemic (Sys) administration, and 2 VE-treated groups, 1 with the same treatment as the Top group with the addition of 5% VE twice a day (Top + VE) and 1 with the same treatment as the Sys group with 5% VE twice a day (SYS + VE). The lens changes were documented with a Scheimpflug camera and changes in light scattering were evaluated quantitatively. The VE-treated groups (Top + VE and Sys + VE) showed a significant inhibition of the increase in the opaque area compared with each of the non-VE-treated groups. The VE ophthalmic solution was strong enough to prevent steroid-induced cataract in rats.

Distributional change of alpha-tocopherol in the rat lens with age.

Concentration of alpha-tocopherol (alpha-Toc.) in the rat lens (1, 4 and 12 months old) was determined in single lenticular layers using gas chromatography and mass spectrometry. The highest concentration of alpha-Toc. in lens was found in the nucleus, followed by the deeper anterior and deeper posterior cortices, the shallow anterior and shallow posterior cortices, and the equatorial region. The topographic alpha-Toc. distribution in the lens did not differ between lenses of 1-, 4- and 12-month-old rats. A significant decrease of alpha-Toc. concentration was seen in lenses of 4- and 12-month-old rats compared to those 1 month old. Concentration and distribution in 4- and 12-month-old rat lenses were almost the same. The concentration of alpha-Toc. in the lens changes in relation to age.

A mild progression type of naphthalene-induced cataract in brown-Norway rats.

Naphthalene-induced rat cataract is a useful experimental cataract--however, because of its short survival period, studies using this model have been for limited purposes. Based on the consideration that the short survival might be caused by systemic toxicity of an overdose of naphthalene administration (1 g/kg body weight every other day), the authors successfully established a naphthalene-induced cataract with mild progression in Brown-Norway rats. The naphthalene administration proposed is to initially administer 0.5 g/kg and after a 1-week interval 1 g/kg of 10% naphthalene once or twice a week through a stomach tube. While the type of lens opacification induced in the two groups administered once and twice a week, respectively, was the same as that seen by the previous administration method, the progression of lens opacification seen in the groups showed a dose-dependent increase. The survival rate in the rats given naphthalene every other day according to the old method was 50% at the 6th week and 0% at the 9th week. Survival of the two new groups was 70 and 60% at the 30th week, respectively. This new type of naphthalene-induced rat cataract should be a suitable model for long-term observations.

[Dynamics of fluoroquinolone agents in tear fluid--a comparison of the dynamic in tears of human and rabbit eyes].

We compared the dynamics of fluoroquinolones administered either systemically or locally, in the tears of human and rabbit eyes. The concentrations of locally administered ofloxacin (OFLX) in the human and rabbit tear fluid were similar. However, there was a difference between the norfloxacin (NFLX) concentrations in human and rabbit tear fluid measured after instillation. The NFLX concentrations in the tear fluid were significantly higher than the OFLX concentrations and they were retained longer with oral administration. On the other hand, the drug dynamics depended on the concentrations of the drug in the serum, and were better in the rabbit eyes than in the human eyes. Knowing the correlation of drug dynamics in rabbit and human eyes may supply useful information about drug dynamics in human eyes, and may be a useful index for clinical treatment.

Differences in naphthalene cataract formation between albino and pigmented rat eyes.

The progression of naphthalene cataracts induced in Brown-Norway rats and Sprague-Dawley rats was compared. The quality of lens changes was basically the same in both strains. However, the cataract progression in Brown-Norway rats showed regularity and was fast as compared with the progression in Sprague-Dawley rats. The cataract development could be divided into three stages. Stage 1: formation of water clefts below the anterior lens capsule (shallow cortex) was observed as the initial change; stage 2: these water clefts extended into the deeper cortical layers, and a semicircular opaque band at the deeper cortical region becomes visible; stage 3: a retroillumination image revealed a ring shadow formation - slit image observation showed wedge-shaped cortical and deeper cortical zonular opacification as the final stage. The expression of these three stages in Sprague-Dawley rats is less uniform and timely delayed as compared with Brown-Norway rats.