Morphometric evaluation of the repair of critical-size defects using demineralized bovine bone and autogenous bone grafts in rat calvaria

Objective: To evaluate the repair of critical-size bone defects in rats treated with demineralized bovine bone (DBB) compared with autogenous bone (AB). Material and method: A bone defect of 8 mm in diameter was created in the calvaria of 50 Rattus norvegicus, treated either with DBB or AB. Sub-groups of five rats of each group were killed at 7, 14, 21, 30 and 90 days post-operatively, and the skulls were removed and processed histologically. Histological sections were stained with hematoxylin and eosin. Result: Histological analysis showed complete closure of the defects with new bone at 90 days in group AB, and substitution of the biomaterial by fibrotic connective tissue in the DBB group at 21 days. Morphometric analysis showed that DBB was rapidly absorbed at 14 days, with its volume density decreasing from 47%+/- 0.8% at 7 days to 1.2%+/- 0.41% at 14 days. Subsequently, volume densities of the connective tissue and neoformed bone increased from 51.1%+/- 11.17% to 86.8%+/- 7.92% and from 1.9%+/- 1.13% to 12%+/- 8.02%, respectively, for the same time interval. The volume density of AB particles did not change throughout the experimental periods, but the amount of new bone increased markedly between 7 and 90 days, from 4.5%+/- 1.57% to 53.5%+/- 6.42% (P < 0.05). Conclusion: DBB did not provide complete repair of the defects, with significantly less new bone formation than in the AB group.

Osteoinductivity potential of rhBMP-2 associated with two carriers in different dosages

The objective of this study was to evaluate bone formation after application of different doses of recombinant human bone morphogenetic protein-2 (rhBMP-2) combined with monoolein or poloxamer gels, in critical bone defects of rats. Forty-five Wistar rats were divided into nine treatment groups with five animals each: I: application of 1 A mu g rhBMP-2 + monoolein; II: 3 A mu g rhBMP-2 + monoolein; III: 7 A mu g rhBMP-2 + monoolein; IV: 1 A mu g rhBMP-2 + poloxamer; V: 3 A mu g rhBMP-2 + poloxamer; VI: 7 A mu g rhBMP-2 + poloxamer; VII: monoolein only; VIII: poloxamer only; and IX: critical bone defect only. A critical-sized defect of 6 mm diameter was produced in the left parietal bone and it was filled with gels of the above mentioned treatments. After 2 weeks, the calvarial bones were removed for histological processing. Bone formation in the groups that received poloxamer gel and rhBMP-2 was not significantly different from the control group (IX). Groups receiving monoolein and rhBMP-2 (1 and 3 A mu g) and those that received only the carriers (VII and VIII) had less bone formation in relation to the control. The association of rhBMP-2 to both poloxamer and monoolein did not exhibit any significant differentiation in bone formation in comparison with the control group.

Serotonergic mechanisms on breathing modulation in the rat locus coeruleus

The locus coeruleus (LC) is a noradrenergic nucleus that plays an important role in the ventilatory response to hypercapnia. This nucleus is densely innervated by serotonergic fibers and contains high density of serotonin (5-HT) receptors, including 5-HT(1A) and 5-HT(2). We assessed the possible modulation of respiratory response to hypercapnia by 5-HT, through 5-HT(1A) and 5-HT(2) receptors, in the LC. To this end, we determined the concentrations of 5-HT and its metabolite 5-hydroxyindole-3-acetic acid (5-HIAA) in the LC after hypercapnic exposure. Pulmonary ventilation (V(E), plethysmograph) was measured before and after unilateral microinjection (100 nL) of WAY-100635 (5-HT(1A) antagonist, 5.6 and 56 mM), 8-OHDPAT (5-HT(1A/7) agonist, 7 and 15 mM), Ketanserin (5-HT(2A) antagonist, 3.7 and 37 mM), or (+/-)-2,5-dimethoxy-4-iodoamphetaminehydrochloride (DOI; 5-HT(2A) agonist, 6.7 and 67 mM) into the LC, followed by a 60-min period of 7% CO(2) exposure. Hypercapnia increased 5-HTIAA levels and 5-HIAA/5-HT ratio within the LC. WAY-100635 and 8-OHDPAT intra-LC decreased the hypercapnic ventilatory response due to a lower tidal volume. Ketanserin increased CO(2) drive to breathing and DOI caused the opposite response, both acting on tidal volume. The current results provide evidence of increased 5-HT release during hypercapnia in the LC and that 5-HT presents an inhibitory modulation of the stimulatory role of LC on hypercapnic ventilatory response, acting through postsynaptic 5-HT(2A) receptors in this nucleus. In addition, hypercapnic responses seem to be also regulated by presynaptic 5-HT(1A) receptors in the LC.

Effect of Sodium Cyclamate on the Rat Fetal Exocrine Pancreas: a Karyometric and Stereological Study

The cyclamate, a sweetner substance derived from N-cyclo-hexyl-sulfamic acid, is largely utilized as a non-caloric artificial edulcorant in foods and beverages as well as in the pharmaceutical industry. The objective of this study was to evaluate karyometric and stereological alterations in the rat fetal pancreas resulting from the intraperitoneal administration of sodium cyclamate. The exocrine pancreas of ten fetuses of rats were evaluated, five treated and five controls chosen at random, in which five rats that received from the 10th to 14th days of pregnancy an intraperitoneal daily injection of sodium cyclamate at 60 mg/Kg of body weight during 5 days. At the 20th day of gestation, the animals were removed and weighed, as were their placentas; the length of the umbilical cords also were measured. After the laboratory processing, semi-seriated 6mm cuts stained with haematoxyline and cosine were performed. In seven karyometric parameters (major, minor, and medium diameters, volume, area, perimeter, and volume-area ratio), the increase was statistically significant in the treated group when compared with control group. Stereological parameters showed in the treated group a significant increase in the cellular volume and a significant reduction in the numerical cellular density. These results showed that the sodium cyclamate in pregnant rats led to retardation of fetal development and hypertrophy in the exocrine pancreas of the rat fetuses.

Long-term effects of developmental exposure to di-n-butyl-phthalate (DBP) on rat prostate: Proliferative and inflammatory disorders and a possible role of androgens

In the present study we evaluated the toxic effects on the male adult rat prostate of DBP exposure during fetal and lactational periods, because although many studies have addressed the influence of phthalates on the male reproductive system, only a few have discussed their possible effects on prostate development. Pregnant females were distributed into two experimental groups: Control (C) and Treated (T). The females of the T group received DBP (100 mg/kg, by gavage) from gestation day 12 to postnatal day 21, while C rats received the vehicle (corn oil). In adulthood (90 days old), the animals were euthanized. The serum and testicular testosterone levels were measured. Ventral prostate was removed and weighed. Distal segment fragments of the ventral prostate were fixed and processed for histochemistry and immunohistochemistry to detect androgen receptor (AR) and Ki67 antigens. Protein extraction from ventral prostate fragments was performed for AR immunoblotting and Gelatin zymography for MMP-2 and MMP-9 (MMP, metalloproteinase). Stereological and histopathological analyses were also performed. Serum and testicular testosterone levels and prostate weight were comparable between groups. In the T group the relative proportions (%) of epithelial (C=32.86; T=42.04*) and stromal (C=21.61; T=27.88*) compartments were increased, while the luminal compartment was decreased (C=45.54; T=30.08*), *p < 0.05. In T, disseminated inflammatory infiltrate in the stroma, associated or not with epithelial dysplasia and PIN (Prostatic Intraepithelial Neoplasia), was observed. Increases in AR expression, proliferation index and metalloproteinase 9 (MMP-9) activity were noted in T animals. In some T animals, collagen fibrils accumulated adjacent to the epithelium. As far as we are aware, this is the first report in the literature showing that phthalates could play a role in proliferative and inflammatory disorders of the rat prostate. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Effects of short-term acetaminophen and celecoxib treatment on orthodontic tooth movement and neuronal activation in rat

Non-steroidal anti-inflammatory drugs (NSAIDs) have been used for pain relief in orthodontics, but clinical studies reported that they may reduce tooth movement (TM). By other side, TM seems to activate brain structures related to nociception, but the effects of NSAIDs in this activation have not been studied yet. We analyzed the effect of short-term treatment with acetaminophen or celecoxib in the separation of rat upper incisors, as well as in neuronal activation of the spinal trigeminal nucleus, following tooth movement. Thirty rats (400-420 g) were pretreated through oral gavage (1 ml/dose)with acetaminophen (200 mg/kg), celecoxib (50 mg/kg) or vehicle (carboxymethylcellulose 0.4%). After 30 min, they received an activated (30 g) orthodontic appliance for TM. In controls, this appliance was immediately removed after its introduction. Rats received ground food, and every 12 h, one of the drugs or vehicle. After 48 h, they were anesthetized, maxilla was radiographed, and were perfused with 4% paraformaldehyde. Brains were further processed for Fos immunohistochemistry. TM induced incisor distalization (p < 0.05) and neuronal activation of the spinal trigeminal nucleus. Treatment with both drugs did not affect tooth movement, but reduced c-fos expression in the caudalis subnucleus. No changes in c-fos expression were seen in the oralis and interpolaris subnuclei. We conclude that neither celecoxib nor acetaminophen seems to affect tooth movement, when used for 2 days, but both drugs are able to reduce the activation of brain structures related to nociception. Short-term treatment with celecoxib, thus, may be a therapeutic alternative to acetaminophen when the latter is contra indicated. (C) 2009 Elsevier Inc. All rights reserved.

Eag1 potassium channel immunohistochemistry in the CNS of adult rat and selected regions of human brain

Eag1 (K(v)10.1) is the founding member of an evolutionarily conserved superfamily of voltage-gated K+ channels. In rats and humans Eag1 is preferentially expressed in adult brain but its regional distribution has only been studied at mRNA level and only in the rat at high resolution. The main aim of the present study is to describe the distribution of Eag1 protein in adult rat brain in comparison to selected regions of the human adult brain. The distribution of Eag1 protein was assessed using alkaline-phosphatase based immunohistochemistry. Eag1 immunoreactivity was widespread, although selective, throughout rat brain, especially noticeable in the perinuclear space of cells and proximal regions of the extensions, both in rat and human brain. To relate the results to the relative abundance of Eag1 transcripts in different regions of rat brain a reverse-transcription coupled to quantitative polymerase chain reaction (real time PCR) was performed. This real time PCR analysis showed high Eag1 expression in the olfactory bulb, cerebral cortex, hippocampus, hypothalamus, and cerebellum. The results indicate that Eag1 protein expression greatly overlaps with mRNA distribution in rats and humans. The physiological relevance of potassium channels in the different regions expressing Eag1 protein is discussed. (C) 2008 IBRO. Published by Elsevier Ltd. All rights reserved.

Effects of a mixture of growth factors and proteins on the development of the osteogenic phenotype in human alveolar bone cell cultures

Strategies to promote bone repair have included exposure of cells to growth factor (GF) preparations from blood that generally include proteins as part of a complex mixture. This study aimed to evaluate the effects of such a mixture on different parameters of the development of the osteogenic phenotype in vitro. Osteoblastic cells were obtained by enzymatic digestion of human alveolar bone and cultured under standard osteogenic conditions until subconfluence. They were subcultured on Thermanox coverslips up to 14 days. Treated cultures were exposed during the first 7 days to osteogenic medium supplemented with a GFs + proteins mixture containing the major components found in platelet extracts [plate I et-derived growth factor-BB, transforming growth factor (TGF)-beta 1, TGF-beta 2, albumin, fibronectin, and thrombospondin] and to osteogenic medium alone thereafter. Control cultures were exposed only to the osteogenic medium. Treated cultures exhibited a significantly higher number of adherent cells from day 4 onward and of cycling cells at days 1 and 4, weak alkaline phosphatase (ALP) labeling, and significantly decreased levels of ALP activity and mRNA expression. At day 14, no Alizarin red-stained nodular areas were detected in cultures treated with GFs + proteins. Results were confirmed in the rat calvaria-derived osteogenic cell culture model. The addition of bone morphogenetic protein 7 or growth and differentiation factor 5 to treated cultures upregulated Runx2 and ALP mRNA expression, but surprisingly, ALP activity was not restored. These results showed that a mixture of GFs + proteins affects the development of the osteogenic phenotype both in human and rat cultures, leading to an increase in the number of cells, but expressed a less differentiated state.

A nitric oxide synthase inhibitor decreases 6-hydroxydopamine effects on tyrosine hydroxylase and neuronal nitric oxide synthase in the rat nigrostriatal pathway

There is evidence that nitric oxide plays a role in the neurotransmitter balance within the basal ganglia and in the pathology of Parkinson`s disease. In the present work we investigated in striatal 6-hydroxydopamine (6-OHDA) lesioned rats the effects of a nitric oxide synthase (NOS) inhibitor, NG-nitro-L-arginine (L-NOARG), given systemically on both the dopaminergic (DA) neuronal loss and the neuronal NOS cell density. We analyzed the DA neuronal loss through tyrosine hydroxylase immunohistochemistry (TH). The nitrergic system was evaluated using an antibody against the neuronal NOS (nNOS) isoform. Treatment with the L-NOARG significantly reduced 6-OHDA-induced dopaminergic damage in the dorsal striatum, ventral substantia nigra and lateral globus pallidus, but had no effects in the dorsal substantia nigra and in the cingulate cortex. Furthermore, L-NOARG reduced 6-OHDA-induced striatal increase, and substantia nigra compacta decrease, in the density of neuronal nitric oxide synthase positive cells. These results suggest that nitric oxide synthase inhibition may decrease the toxic effects of 6-OHDA on dopaminergic terminals and on dopamine cell bodies in sub-regions of the SN and on neuronal nitric oxide synthase cell density in the rat brain. (c) 2008 Elsevier B.V. All rights reserved.

Assessment of the biocompatibility of Epiphany root canal sealer in rat subcutaneous tissues

Objective. The aim of this study was to evaluate the biocompatibility of the root canal sealer Epiphany in rat subcutaneous tissues. Study design. Polyethylene tubes were filled with the sealer (I: Epiphany; II: photoactivated Epiphany; III: Epiphany associated with self-etch primer; IV: photoactivated Epiphany associated with primer; and V: control group) and later implanted into 4 different regions of the dorsum of 15 adult male rats (Rattus novergicus, Albinus Wistar). After 7, 21, and 42 days, 5 animals were killed, obtaining 4 samples per group, in addition to the control group, at each analyzed time. Results. In all periods, Epiphany induced a mild inflammatory reaction. However, in group II, in which the primer was not used, extensive necrosis and a moderate to intense inflammatory reaction were observed, mainly after 7 and 21 days. Conclusion. Epiphany sealer appears biocompatible when tested on rat subcutaneous tissues.

Projections from the substantia nigra pars reticulata to the motor thalamus of the rat: Single axon reconstructions and immunohistochemical study

This is a study in the rat of the distribution of specific neurotransmitters in neurones projecting from the substantia nigra reticulata (SNR) to the ventrolateral (VL) and ventromedial (VM) thalamic nuclei. Individual axons projecting from the SNR to these thalamic nuclei have also been reconstructed following small injection of the anterograde tracer dextran biotin into the the SNR. Analysis of reconstructions revealed two populations of SNR neurones projecting onto the VL and VM thalamic nuclei. One group projects directly onto the VM and VL, and the other projects to the VM/VL and to the parafascicular nucleus. In another set of experiments Fluoro-Gold was injected into the VL/VM to label SNR projection neurones retrogradely, and immunohistochemistry was performed to determine the distribution of choline acetyltransferase (ChAT), vesicular acetylcholine transporter (VAChT), gamma -aminobutyric acid (GABA), and glutamate in Fluoro-Gold-labelled SNR projection neurones. Most SNR-VL/VM thalamic projection neurones were immunoreactive to acetylcholine or glutamate, whereas only 25% of the projection neurones were found to be immunoreactive to GABA. (C) 2001 Wiley-Liss, Inc.

Cloning and function of the rat colonic epithelial K+ channel K(V)LQT1

K(V)LQT1 (K(V)LQ1) is a voltage-gated K+ channel essential for repolarization of the heart action potential that is defective in cardiac arrhythmia. The channel is inhibited by the chromanol 293B, a compound that blocks cAMP-dependent electrolyte secretion in rat and human colon, therefore suggesting expression of a similar type of K+ channel in the colonic epithelium. We now report cloning and expression of K(V)LQT1 from rat colon. Overlapping clones identified by cDNA-library screening were combined to a full length cDNA that shares high sequence homology to K(V)LQT1 cloned from other species. RT-PCR analysis of rat colonic musoca demonstrated expression of K(V)LQT1 in crypt cells and surface epithelium. Expression of rK(V)LQT1 in Xenopus oocytes induced a typical delayed activated K+ current. that was further activated by increase of intracellular cAMP but not Ca2+ and that was blocked by the chromanol 293B. The same compound blocked a basolateral cAMP-activated K+ conductance in the colonic mucosal epithelium and inhibited whole cell K+ currents in patch-clamp experiments on isolated colonic crypts. We conclude that K(V)QT1 is forming an important component of the basolateral cAMP-activated K+ conductance in the colonic epithelium and plays a crucial role in diseases like secretory diarrhea and cystic fibrosis.

Structure-hepatic disposition relationships for cationic drugs in isolated perfused rat livers: Transmembrane exchange and cytoplasmic binding process

This work studied the structure-hepatic disposition relationships for cationic drugs of varying lipophilicity using a single-pass, in situ rat liver preparation. The lipophilicity among the cationic drugs studied in this work is in the following order: diltiazem. propranolol. labetalol. prazosin. antipyrine. atenolol. Parameters characterizing the hepatic distribution and elimination kinetics of the drugs were estimated using the multiple indicator dilution method. The kinetic model used to describe drug transport (the two-phase stochastic model) integrated cytoplasmic binding kinetics and belongs to the class of barrier-limited and space-distributed liver models. Hepatic extraction ratio (E) (0.30-0.92) increased with lipophilicity. The intracellular binding rate constant (k(on)) and the equilibrium amount ratios characterizing the slowly and rapidly equilibrating binding sites (K-S and K-R) increase with the lipophilicity of drug (k(on) : 0.05-0.35 s(-1); K-S : 0.61-16.67; K-R : 0.36-0.95), whereas the intracellular unbinding rate constant (k(off)) decreases with the lipophilicity of drug (0.081-0.021 s(-1)). The partition ratio of influx (k(in)) and efflux rate constant (k(out)), k(in)/k(out), increases with increasing pK(a) value of the drug [from 1.72 for antipyrine (pK(a) = 1.45) to 9.76 for propranolol (pK(a) = 9.45)], the differences in k(in/kout) for the different drugs mainly arising from ion trapping in the mitochondria and lysosomes. The value of intrinsic elimination clearance (CLint), permeation clearance (CLpT), and permeability-surface area product (PS) all increase with the lipophilicity of drug [CLint (ml . min(-1) . g(-1) of liver): 10.08-67.41; CLpT (ml . min(-1) . g(-1) of liver): 10.80-5.35; PS (ml . min(-1) . g(-1) of liver): 14.59-90.54]. It is concluded that cationic drug kinetics in the liver can be modeled using models that integrate the presence of cytoplasmic binding, a hepatocyte barrier, and a vascular transit density function.

Probing the effect of vehicles on topical delivery: Understanding the basic relationship between solvent and solute penetration using silicone membranes

Purpose. In the present study we examined the relationship between solvent uptake into a model membrane (silicone) with the physical properties of the solvents (e.g., solubility parameter, melting point, molecular weight) and its potential predictability. We then assessed the subsequent topical penetration and retention kinetics of hydrocortisone from various solvents to define whether modifications to either solute diffusivity or partitioning were dominant in increasing permeability through solvent-modified membranes. Methods. Membrane sorption of solvents was determined from weight differences following immersion in individual solvents, corrected for differences in density. Permeability and retention kinetics of H-3-hydrocortisone, applied as saturated solutions in the various solvents, were determined over 48 h in horizontal Franz-type glass diffusion cells. Results. Solvent sorption into the membrane could be related to differences in solubility parameters, MW and hydrogen bonding (r(2) = 0.76). The actual and predicted volume of solvent sorbed into the membrane was also found to be linearly related to Log hydrocortisone flux, with changes in both diffusivity and partitioning of hydrocortisone observed for the different solvent vehicles. Conclusions. A simple structure-based predictive model can be applied to the sorption of solvents into silicone membranes. Changes in solute diffusivity and partitioning appeared to contribute to the increased hydrocortisone flux observed with the various solvent vehicles. The application of this predictive model to the more complex skin membrane remains to be determined.