Rat subcutaneous tissue response to MTA Fillapex® and Portland cement

The aim of this study was to evaluate the response of rat subcutaneous tissue to MTA Fillapex® (Angelus), an experimental root canal filling material based on Portland cement and propylene glycol (PCPG), and a zinc oxide, eugenol and iodoform (ZOEI) paste. These materials were placed in polyethylene tubes and implanted into the dorsal connective tissue of Wistar rats for 7 and 15 days. The specimens were stained with hematoxylin and eosin, and evaluated regarding inflammatory reaction parameters by optical microscopy. The intensity of inflammatory response against the sealers was analyzed by two blinded and previously calibrated examiners for all experimental periods (kappa=0.96). The histological evaluation showed that all materials caused a moderate inflammatory reaction at 7 days, which subsided with time. A greater inflammatory reaction was observed at 7 days in the tubes filled with ZOEI paste. Tubes filled with MTA Fillapex presented some giant cells, macrophages and lymphocytes after 7 days. At 15 days, the presence of fibroblasts and collagen fibers was observed indicating normal tissue healing. The tubes filled with PCPG showed similar results to those observed in MTA Fillapex. At 15 days, the inflammatory reaction was almost absent at the tissue, with several collagen fibers indicating normal tissue healing. Data were analyzed by the nonparametric Kruskal-Wallis test (?=0.05). Statistically significant difference (p<0.05) was found only between PCPG at 15 days and ZOEI at 7 days groups. No significant differences were observed among the other groups/periods (p>0.05). MTA Fillapex and Portland cement added with propylene glycol had greater tissue compatibility than the PCPG paste.

Chitosan-based biomaterials used in critical-size bone defects: radiographic study in rat's calvaria

OBJETIVO: Este estudo avaliou através de imagens radiográficas digitais, a ação de biomateriais de quitosana e de cloridrato de quitosana, com baixo e alto peso molecular, utilizados na correção de defeitos ósseos de tamanho crítico (DOTC)em calvária de ratos. MATERIAL E MÉTODO: DOTCs com 8 mm de diâmetro foram criados cirurgicamente na calvária de 50 ratos Holtzman. Em 10 animais o defeito foi preenchido foram preenchidos com coágulo sanguíneo (controle negativo). Os 40 animais restantes foram divididos de acordo com o biomaterial utilizado no preenchimento do defeito (quitosana de baixo peso e de alto peso molecular, e cloridrato de quitosana de baixo e de alto peso molecular), e foram avaliados em dois períodos experimentais (15 e 60 dias), totalizando 5 animais/biomaterial/período de avaliação. RESULTADO: A avaliação radiográfica foi feita utilizando duas radiografias digitais do crânio do animal: uma tomada logo após o defeito ósseo ser criado e a outra no momento do sacrifício. Nessas imagens, foi avaliada a densidade óssea radiográfica inicial e a final na área do defeito, que foram comparadas. As análises na densidade óssea radiográfica indicaram aumento da densidade óssea radiográfica dos DOTCs tratados para todos os biomateriais testados, em ambos os períodos. Resultados semelhantes foram encontrados no grupo controle. CONCLUSÃO: Conclui-se que os biomateriais de quitosana testados não foram capazes de aumentar a densidade radiográfica em DOTC realizados em calvária de ratos.

Suppressive effect of low-level laser therapy on tracheal hyperresponsiveness and lung inflammation in rat subjected to intestinal ischemia and reperfusion

Intestinal ischemia and reperfusion (i-I/R) is an insult associated with acute respiratory distress syndrome (ARDS). It is not known if pro- and anti-inflammatory mediators in ARDS induced by i-I/R can be controlled by low-level laser therapy (LLLT). This study was designed to evaluate the effect of LLLT on tracheal cholinergic reactivity dysfunction and the release of inflammatory mediators from the lung after i-I/R. Anesthetized rats were subjected to superior mesenteric artery occlusion (45 min) and killed after clamp release and preestablished periods of intestinal reperfusion (30 min, 2 or 4 h). The LLLT (660 nm, 7.5 J/cm(2)) was carried out by irradiating the rats on the skin over the right upper bronchus for 15 and 30 min after initiating reperfusion and then euthanizing them 30 min, 2, or 4 h later. Lung edema was measured by the Evans blue extravasation technique, and pulmonary neutrophils were determined by myeloperoxidase (MPO) activity. Pulmonary tumor necrosis factor-α (TNF-α), interleukin-10 (IL-10), intercellular adhesion molecule-1 (ICAM-1), and isoform of NO synthase (iNOS) mRNA expression were analyzed by real-time PCR. TNF-α, IL-10, and iNOS proteins in the lung were measured by the enzyme-linked immunoassay technique. LLLT (660 nm, 7.5 J/cm(2)) restored the tracheal hyperresponsiveness and hyporesponsiveness in all the periods after intestinal reperfusion. Although LLLT reduced edema and MPO activity, it did not do so in all the postreperfusion periods. It was also observed with the ICAM-1 expression. In addition to reducing both TNF-α and iNOS, LLLT increased IL-10 in the lungs of animals subjected to i-I/R. The results indicate that LLLT can control the lung's inflammatory response and the airway reactivity dysfunction by simultaneously reducing both TNF-α and iNOS.

Inflammatory responses in the rat superior colliculus after eye enucleation

Ocular enucleation induces profound morphological alterations in central visual areas. However, little is known about the response of glial cells and possible inflammatory processes in visual brain areas resulting from eye enucleation. In this study, immunoblotting and immunostaining assays revealed increased expression of astrocyte and microglia markers in the rat superior colliculus (SC) between 1 and 15 days after contralateral enucleation. A transient increase of neuronal COX-2 protein expression was also found in the SC. To evaluate the role of an anti-inflammatory drug in attenuating both COX-2 and glial cell activation, the synthetic glucocorticoid dexamethasone (DEX) was administered (1mg/kg i.p., for 3 days) to enucleated rats. Immunoblotting data revealed that DEX treatment significantly inhibited COX-2 protein expression. Postlesion immunostaining for astrocyte and microglia markers was also significantly reduced by DEX treatment. These findings suggest that the removal of retinal ganglion cell input generates inflammatory responses in central retinorecipient structures

Analysis of new bone formation induced by periosteal distraction in a rat calvarium model

Background: A controlled, gradual distraction of the periosteum is expected to result in the formation of new bone. Purpose: This study was designed to estimate the possibility of new bone formation by periosteal distraction in a rat calvarium model. Material and Methods: Sixteen animals were subjected to a 7-day latency period and distraction rate at 0.4 mm/24 hours for 10 days. Two experimental groups with seven rats each were killed at 10 and 20 days of consolidation period and analyzed by means of microcomputed tomography, histologically and histomorphometry. Results: In the central regions underneath the disk device, signs of both bone apposition and bone resorption were observed. Peripheral to the disc, new bone was consistently observed. This new bone was up to two and three times thicker than the original bone after a 10- and 20-day consolidation period, respectively. Signs of ongoing woven bone formation indicated that the stimulus for new bone formation was still present. There were no statistically significant differences regarding bone density, bone volume, and total bone height between the two groups. Conclusion: The periosteal distraction model in the rat calvarium can stimulate the formation of considerable amounts of new bone.

Functional Imaging of Pheochromocytoma with Ga-DOTATOC and C-HED in a Genetically Defined Rat Model of Multiple Endocrine Neoplasia

Rats affected by the MENX multitumor syndrome develop pheochromocytoma (100%). Pheochromocytomas are uncommon tumors and animal models are scarce, hence the interest in MENX rats to identify and preclinically evaluate novel targeted therapies. A prerequisite for such studies is a sensitive and noninvasive detection of MENXassociated pheochromocytoma. We performed positron emission tomography (PET) to determine whether rat pheochromocytomas are detected by tracers used in clinical practice, such as 68Ga-DOTATOC (somatostatin analogue) or (11)C-Hydroxyephedrine (HED), a norepinephrine analogue. We analyzed four affected and three unaffected rats. The PET scan findings were correlated to histopathology and immunophenotype of the tumors, their proliferative index, and the expression of genes coding for somatostatin receptors or the norepinephrine transporter. We observed that mean 68Ga-DOTATOC standard uptake value (SUV) in adrenals of affected animals was 23.3 ± 3.9, significantly higher than in control rats (15.4 ± 7.9; P = .03). The increase in mean tumor-to-liver ratio of (11)C-HED in the MENX-affected animals (1.6 ± 0.5) compared to controls (0.7 ± 0.1) was even more significant (P = .0016). In a unique animal model, functional imaging depicting two pathways important in pheochromocytoma biology discriminated affected animals from controls, thus providing the basis for future preclinical work with MENX rats.

Relative Contributions of Osteogenic Tissues to New Bone Formation in Periosteal Distraction Osteogenesis: Histological and Histomorphometrical Evaluation in a Rat Calvaria

Background: The relative contributions of different, potential factors to new bone formation in periosteal distraction osteogenesis are unknown. Purpose: The aim of the present study was to assess the influence of original bone and periosteum on bone formation during periosteal distraction osteogenesis in a rat calvarial model by means of histology and histomorphometry. Methods: A total of 48 rats were used for the experiment. The contribution of the periosteum was assessed by either intact or incised periosteum or an occlusive versus a perforated distraction plate. The cortical bone was either left intact or perforated. Animals were divided in eight experimental groups considering the three possible treatment modalities. All animals were subjected to a 7-day latency period, a 10-day distraction period and a 7-day consolidation period. The newly formed bone was analyzed histologically and histomorphometrically. Results: New, mainly woven bone was found in all groups. Differences in the maximum height of new bone were observed and depended on location. Under the distraction plate, statistically significant differences in maximum bone height were found between the group with perforations in both cortical bone and distraction plate and the group without such perforations. Conclusions: If the marrow cavities were not opened, the contribution to new bone formation was dominant from the periosteum. If the bone perforations opened the marrow cavities, a significant contribution to new bone formation originated from the native bone.

Adjunctive daptomycin attenuates brain damage and hearing loss more efficiently than rifampin in infant rat pneumococcal meningitis

Exacerbation of cerebrospinal fluid (CSF) inflammation in response to bacteriolysis by beta-lactam antibiotics contributes to brain damage and neurological sequelae in bacterial meningitis. Daptomycin, a nonlytic antibiotic acting on Gram-positive bacteria, lessens inflammation and brain injury compared to ceftriaxone. With a view to a clinical application for pediatric bacterial meningitis, we investigated the effect of combining daptomycin or rifampin with ceftriaxone in an infant rat pneumococcal meningitis model. Eleven-day-old Wistar rats with pneumococcal meningitis were randomized to treatment starting at 18 h after infection with (i) ceftriaxone (100 mg/kg of body weight, subcutaneously [s.c.], twice a day [b.i.d.]), (ii) daptomycin (10 mg/kg, s.c., daily) followed 15 min later by ceftriaxone, or (iii) rifampin (20 mg/kg, intraperitoneally [i.p.], b.i.d.) followed 15 min later by ceftriaxone. CSF was sampled at 6 and 22 h after the initiation of therapy and was assessed for concentrations of defined chemokines and cytokines. Brain damage was quantified by histomorphometry at 40 h after infection and hearing loss was assessed at 3 weeks after infection. Daptomycin plus ceftriaxone versus ceftriaxone significantly (P < 0.04) lowered CSF concentrations of monocyte chemoattractant protein 1 (MCP-1), MIP-1α, and interleukin 6 (IL-6) at 6 h and MIP-1α, IL-6, and IL-10 at 22 h after initiation of therapy, led to significantly (P < 0.01) less apoptosis, and significantly (P < 0.01) improved hearing capacity. While rifampin plus ceftriaxone versus ceftriaxone also led to lower CSF inflammation (P < 0.02 for IL-6 at 6 h), it had no significant effect on apoptosis and hearing capacity. Adjuvant daptomycin could therefore offer added benefits for the treatment of pediatric pneumococcal meningitis.

Cardiac transplantation with hearts from donors after circulatory declaration of death: haemodynamic and biochemical parameters at procurement predict recovery following cardioplegic storage in a rat model

OBJECTIVES: Donation after circulatory declaration of death (DCDD) could significantly improve the number of cardiac grafts for transplantation. Graft evaluation is particularly important in the setting of DCDD given that conditions of cardio-circulatory arrest and warm ischaemia differ, leading to variable tissue injury. The aim of this study was to identify, at the time of heart procurement, means to predict contractile recovery following cardioplegic storage and reperfusion using an isolated rat heart model. Identification of reliable approaches to evaluate cardiac grafts is key in the development of protocols for heart transplantation with DCDD. METHODS: Hearts isolated from anaesthetized male Wistar rats (n = 34) were exposed to various perfusion protocols. To simulate DCDD conditions, rats were exsanguinated and maintained at 37°C for 15-25 min (warm ischaemia). Isolated hearts were perfused with modified Krebs-Henseleit buffer for 10 min (unloaded), arrested with cardioplegia, stored for 3 h at 4°C and then reperfused for 120 min (unloaded for 60 min, then loaded for 60 min). Left ventricular (LV) function was assessed using an intraventricular micro-tip pressure catheter. Statistical significance was determined using the non-parametric Spearman rho correlation analysis. RESULTS: After 120 min of reperfusion, recovery of LV work measured as developed pressure (DP)-heart rate (HR) product ranged from 0 to 15 ± 6.1 mmHg beats min(-1) 10(-3) following warm ischaemia of 15-25 min. Several haemodynamic parameters measured during early, unloaded perfusion at the time of heart procurement, including HR and the peak systolic pressure-HR product, correlated significantly with contractile recovery after cardioplegic storage and 120 min of reperfusion (P < 0.001). Coronary flow, oxygen consumption and lactate dehydrogenase release also correlated significantly with contractile recovery following cardioplegic storage and 120 min of reperfusion (P < 0.05). CONCLUSIONS: Haemodynamic and biochemical parameters measured at the time of organ procurement could serve as predictive indicators of contractile recovery. We believe that evaluation of graft suitability is feasible prior to transplantation with DCDD, and may, consequently, increase donor heart availability.

Lactate, not glucose, up-regulates mitochondrial oxygen consumption both in sham and lateral fluid percussed rat brains

OBJECTIVE: Failure of energy metabolism after traumatic brain injury may be a major factor limiting outcome. Although glucose is the primary metabolic substrate in the healthy brain, the well documented surge in tissue lactate after traumatic brain injury suggests that lactate may provide an energy need that cannot be met by glucose. We hypothesized, therefore, that administration of lactate or the combination of lactate and supraphysiological oxygen may improve mitochondrial oxidative respiration in the brain after rat fluid percussion injury. We measured oxygen consumption (VO2) to determine what effects glucose, lactate, oxygen, and the combination of lactate and oxygen have on mitochondrial respiration in both injured and uninjured rat brain tissue. METHODS: Anesthetized Sprague-Dawley rats were intubated and ventilated with either 0.21 or 1.0 fraction of inspired oxygen (FIO2). Brain tissue from acute sham animals was subjected in vitro to 1.1 mM, 12 mM and 100 mM concentrations of glucose and L-lactate. In another group, injury (fluid percussion injury of 2.5 +/- 0.02 atmospheres) was induced over the left hemisphere. The VO2 of mug amounts of brain tissues were measured in a microrespirometry system (Cartesian diver). RESULTS: The VO2 was found to be independent of glucose concentrations, but dose-dependent for lactate. Moreover, the lactate dependent VO2s were all significantly higher than those generated by glucose. Injured rats on FIO2 0.21 had brain tissue VO2 rates that were significantly lower than those of shams or preinjury levels. In injured rats treated with FIO2 1.0, the reduction in VO2 levels was prevented. Injured rats that received an intravenous infusion of 100 mM lactate had VO2 rates that were significantly higher than those obtained with FIO2 1.0. Combined treatment further boosted the lactate generated VO2 rates by approximately 15%. CONCLUSION: Glucose sustains mitochondrial respiration at a low level "fixed" rate because, despite increasing its concentration nearly 100-fold, it cannot up-regulate VO2 after fluid percussion injury. Lactate produces a dose-dependent VO2 response, possibly enabling mitochondria to meet the increased energy needs of the injured brain.

Fluid percussion injury transiently increases then decreases brain oxygen consumption in the rat

The oxygen consumption (VO2 microL/h/mg) of sham and of traumatized rat brains within 30 min and 6 h after a lateral fluid percussion injury (FPI) was measured with the Cartesian microrespirometer. Brain slices were cut at the plain of injury and site-specific 20-60-microg cores of tissue were transferred to the microrespirometer. In sham brains, the cortical VO2 (CVO2) was 13.78+/-0.64 and the hippocampal VO2 (HPVO2) was 11.20+/-0.58 microL/h/mg (p<0.05). Within 30 min of the injury, the respective values of 16.89+/-0.55 and 14.91+/-0.06 were significantly increased (p<0.05). The combined VO2 (CVO2, HPVO2) of 12.49+/-0.06 microL/h/mg in shams was significantly less than the combined VO2 of 15.90+/-0.59 microL/h/mg at 30 min post FPI (p<0.001). The maximal CVO2 of 19.49+/-1.10 microL/h/mg and the maximal HPVO2 of 15.98+/-0.99 microL/h/mg were both obtained from the ipsilateral side of the injury. Whereas the contralateral cortical value for injured brains was not significantly different from that of the shams, both ipsilateral and contralateral hippocampal values were significantly greater than that of the shams in response to injury (p<0.05). By 6 h postinjury, the combined VO2 had dropped to 10.01+/-0.84 microL/h/mg but was not significantly lower than the sham values. The data indicate that normal CVO2 is greater than normal HPVO2. The FPI produces significant increases in both CVO2 and HPVO2. Also, while the immediate increase in CVO2 appears to be injury-site dependent, that is, regional, the increase in HPVO2 appears to be global.

Spindle burst like spike discharge oscillations in organotypic cultures of neonatal rat cortex

Early network oscillations and spindle bursts are typical patterns of spontaneous rhythmic activity in cortical networks of neonatal rodents in vivo and in vitro. The latter can also be triggered in vivo by stimulation of afferent inputs. The mechanisms underlying such oscillations undergo profound developmental changes in the first postnatal weeks. Their possible role in cortical development is postulated but not known in detail. We have studied spontaneous and evoked patterns of activity in organotypic cultures of slices from neonatal rat cortex grown on multielectrode arrays (MEAs) for extracellular single- and multi-unit recording. Episodes of spontaneous spike discharge oscillations at 7 - 25 Hz lasting for 0.6 - 3 seconds appeared in about half of these cultures spontaneously and could be triggered by electrical stimulation of few distinct electrodes. These oscillations usually covered only restricted areas of the slices. Besides oscillations, single population bursts that spread in a wavelike manner over the whole slice also appeared spontaneously and were triggered by electrical stimulation. In most but not all cultures, population bursts preceded the oscillations. Both population bursts and spike discharge oscillations required intact glutamatergic synaptic transmission since they were suppressed by the AMPA/kainate glutamate receptor antagonist CNQX. The NMDA antagonist d-APV suppressed the oscillations but not the population bursts, suggesting an involvement of NMDA receptors in the oscillations. These findings show that spindle burst like cortical rhythms are reproduced in organotypic cultures of neonatal cortex. The culture model thus allows investigating the role of such rhythms in cortical circuit formation. Supported by SNF grant No. 3100A0-107641/1.

Dextran sulfate facilitates anti-CD4 mAb-induced long-term rat cardiac allograft survival after prolonged cold ischemia

Ischemia/reperfusion injury leads to activation of graft endothelial cells (EC), boosting antigraft immunity and impeding tolerance induction. We hypothesized that the complement inhibitor and EC-protectant dextran sulfate (DXS, MW 5000) facilitates long-term graft survival induced by non-depleting anti-CD4 mAb (RIB 5/2). Hearts from DA donor rats were heterotopically transplanted into Lewis recipients treated with RIB 5/2 (20 mg/kg, days-1,0,1,2,3; i.p.) with or without DXS (grafts perfused with 25 mg, recipients treated i.v. with 25 mg/kg on days 1,3 and 12.5 mg/kg on days 5,7,9,11,13,15). Cold graft ischemia time was 20 min or 12 h. Median survival time (MST) was comparable between RIB 5/2 and RIB 5/2+DXS-treated recipients in the 20-min group with >175-day graft survival. In the 12-h group RIB 5/2 only led to chronic rejection (MST = 49.5 days) with elevated alloantibody response, whereas RIB 5/2+DXS induced long-term survival (MST >100 days, p < 0.05) with upregulation of genes related to transplantation tolerance. Analysis of the 12-h group treated with RIB 5/2+DXS at 1-day posttransplantation revealed reduced EC activation, complement deposition and inflammatory cell infiltration. In summary, DXS attenuates I/R-induced acute graft injury and facilitates long-term survival in this clinically relevant transplant model.

Effect of the NMDA-receptor antagonist dextromethorphan in infant rat pneumococcal meningitis

Excitatory amino acids (EAA) and particularly glutamate toxicity have been implicated in the pathogenesis of neuronal injury occurring in bacterial meningitis by activating the N-methyl-d aspartate (NMDA) receptor complex. Here, we evaluated the effect of adjuvant treatment with the antitussive drug dextromethorphan (DM), a non-competitive NMDA receptor antagonist with neuroprotective potential, in an infant rat model of pneumococcal meningitis. The experiments were carried out in postnatal day 6 (P6) and 11 (P11) animals. Pharmacokinetics of DM and its major metabolite dextrorphan (DO) were performed for dose finding. In our study, DM did not alter clinical parameters (clinical score, motor activity, incidence of seizures, spontaneous mortality) and cortical neuronal injury but increased the occurrence of ataxia (P<0.0001). When DM treatment was started at the time of infection (DM i.p. 15 mg/kg at 0, 4, 8 and 16 hours (h) post infection) in P11 animals, an aggravation of apoptotic neuronal death in the hippocampal dentate gyrus was found (P<0.05). When treatment was initiated during acute pneumococcal meningitis (DM i.p. 15 mg/kg at 12 and 15 h and 7.5 mg/kg at 18 and 21 h after infection), DM had no effect on the extent of brain injury but reduced the occurrence of seizures (P<0.03). We conclude that in this infant rat model of pneumococcal meningitis interference of the EEA and NMDA pathway using DM causes ataxia, attenuates epileptic seizures and increases hippocampal apoptosis, but is not effective in protecting the brain from injury.