Subacute Assessment of the Toxicity and Antidepressant-Like Effects of Origanum Majorana L. Polyphenols in Swiss Albino Mice.

Origanum majorana L. is a plant commonly used in folk medicine to treat depression and several neurological disorders. This study aims to evaluate the antidepressant-like effect of the Origanum majorana L. polyphenols (OMP) obtained from the aerial parts using two different depression model tests: The forced swimming test (FST) and the tail suspension test (TST) in Swiss albino mice. The experiments were performed on days 1, 7, 14, and 21 with daily administration of different treatments. Two different doses were chosen for this study (50 and 100 mg/kg), and paroxetine was used as a positive control. Immobility as a consequence of the depression state was significantly reduced following the treatment with OMP, indicating an antidepressant effect. A subacute toxicity study was also performed following the Organization for Economic Co-operation and Development (OECD) Guidelines (407), showing no sign of toxicity for the studied doses. The phytochemical screening revealed the presence of 12 components, all belonging to polyphenols: Arbutin, rosmarinic acid, ursolic acid, quercetin-3-O-glucoside, quercetin-7-O-glucuronic acid, luteolin-7-O-glucoside, kaempferol-3-0-glucuronic acid, Kaempferol-3-0-pentose, caffeic acid, catechin, quercetin, and rutin. These findings suggest that O. majorana has interesting antidepressant-like properties, which deserve further investigation.

Assessment of the rapid and sustained antidepressant-like effects of dextromethorphan in mice.

The glutamatergic system has emerged as a novel pathway for treating major depressive disorder (MDD) with the focus on producing both rapid and sustained antidepressant effects. Dextromethorphan is a noncompetitive N-methyl-d-aspartate (NMDA) receptor antagonist that has produced antidepressant-like effects in forced swim and tail suspension tests (TST); however, the rapid and sustained antidepressant-like effects of dextromethorphan have not been evaluated. This study evaluated the rapid and sustained (24 h) antidepressant-like effects of dextromethorphan (0-32 mg/kg) in C56BL/6 mice using the novelty-induced hypophagia (NIH) test and TST, respectively. Additionally, we evaluated anxiety-related behavior and locomotor effects of dextromethorphan (0-56.0 mg/kg) using the light-dark and open field tests. Dextromethorphan (32 mg/kg) produced acute (30 min) antidepressant-like effects in TST, but failed to produce antidepressant-like effects 24 h after drug administration. Treatment of dextromethorphan (32 mg/kg) alone or in combination with CYP2D6 enzyme inhibitor Quinidine (32 mg/kg) failed to produce rapid antidepressant-like effects by increasing the latency to drink in the NIH test rather than decreasing the latency to drink. Dextromethorphan (56 mg/kg) produced an anxiogenic-like effect by decreasing the time spent in the light side, number of entries, and latency to enter the light side in the light-dark test. Administration of dextromethorphan (0-56 mg/kg) did not significantly alter locomotor activity. Although dextromethorphan is considered a noncompetitive NMDA receptor antagonist, dextromethorphan binds to several monoaminergic receptors (SERT and NET) and likely produces the antidepressant-like effects through these receptors similar to traditional antidepressant drugs. Additionally, these results suggest that the therapeutic window for dextromethorphan in the clinical population is small as similar doses produce antidepressant-like and anxiogenic-like behaviors.

Establishment of an in vivo model for molecular assessment of titanium implant osseointegration in compromised bone.

Shortening of the healing time before loading risks impeding successful titanium implant anchorage into compromised bone. A thorough understanding at the genetic scale of the early phases of bone regeneration at the implant interface is required before the development of strategies to enhance implant osseointegration. In this study a new in vivo implant model to explore the mechanism by which titanium implant osseointegration is affected by the host bone properties is presented. An implant was conceptualized enabling standardized harvesting of peri-implant tissue for quantitative molecular analysis while preserving the mimicking of the clinical setting. The implant is partly indented to provide a well-defined healing compartment from where tissue differentiation and de novo bone formation can be investigated and partly screw-threaded to provide a good implant anchorage into the bone. The feasibility of the implant design was assessed in osteopenic bone conditions, evoked by simulated weightlessness. Wistar rats were either hindlimb unloaded by tail suspension (HU) for 9 days or acted as controls (CTL). The status of compromised bone tissue through 9-days HU was confirmed by micro-X-ray computed tomography. The implant was installed in the proximal tibial bone 7 days after the onset of HU or CTL. Two days postimplantation, the peri-implant regenerating tissue responses were recorded by measuring expression of inflammatory, angiogenic, and bone resorption parameters (hypoxia-inducible factor 1, alpha subunit; vascular endothelial growth factor A; angiopoietin 1; endothelial PAS domain protein 1; fibroblast growth factor 2; tumor necrosis factor; interleukin 11; acid phosphatase 5, tartrate resistant; tumor necrosis factor (ligand) superfamily, member 11/RANKL). We successfully demonstrated that HU-associated bone conditions evoked a significant alteration of expression of the angiogenic markers in the peri-implant regenerative tissue during initial implant osseointegration, whereas the expression levels of the inflammatory and bone resorption parameters remained unchanged. We concluded that this in vivo implant model provides a well-designed and controlled method to examine molecular responses in implant osseointegration to impaired bone conditions. This model may serve to explore the application of anabolic strategies in peri-implant osteogenesis.

Imaging and quantitative assessment of long bone vascularization in the adult rat using microcomputed tomography.

The objective of this study was to develop and validate a technique for both 3D imaging and quantification of the vascular network of bone tissue in the rat. Five month-old male Wistar rats were divided into tail-suspension (21 days) and control groups. Sixty percent barium sulfate solution was infused into the vena cava. The tibiae were evaluated in 2D and 3D before and after decalcification, using conventional microcomputerized tomography (muCT) at 10 and 5 mum resolution and synchrotron radiation (SR) muCT. The perfusion technique and tomography exhibited excellent bone vasculature imaging. Significant positive correlations were observed between 2D histomorphometric and 3D muCT vascular parameters (P < 0.05). 3DmuCT discriminated significant changes of vessel structures in unloading condition: vessel number decreased by 25%, (P < 0.005), vessel separation increased by 27%, P < 0.01. SRmuCT could image sinusoid clusters in bone. muCT is an accurate and reproducible technique for 3D quantitative evaluation of long bone vascularisation in the rat.

Tail suspension increases energy expenditure independently of the melanocortin system in mice.

Space travelers experience anorexia and body weight loss in a microgravity environment, and microgravity-like situations cause changes in hypothalamic activity. Hypothalamic melanocortins play a critical role in the regulation of metabolism. Therefore, we hypothesized that microgravity affects metabolism through alterations in specific hypothalamic signaling pathways, including melanocortin signaling. To address this hypothesis, the microgravity-like situation was produced by an antiorthostatic tail suspension in wild-type and agouti mice, and the effect of tail suspension on energy expenditure and hypothalamic gene expression was examined. Energy expenditure was measured using indirect calorimetry before and during the tail suspension protocol. Hypothalamic tissues were collected for gene expression analysis at the end of the 3 h tail suspension period. Tail suspension significantly increased oxygen consumption, carbon dioxide production, and heat production in wild-type mice. Tail suspension-induced increases in energy expenditure were not attenuated in agouti mice. Although tail suspension did not alter hypothalamic proopiomelanocortin (POMC) and agouti-related protein (AGRP) mRNA levels, it significantly increased hypothalamic interleukin 6 (Il-6) mRNA levels. These data are consistent with the hypothesis that microgravity increases energy expenditure and suggest that these effects are mediated through hypothalamic signaling pathways that are independent of melanocortins, but possibly used by Il-6.

A DIGE approach for the assessment of rat soleus muscle changes during unloading: effect of acetyl-L-carnitine supplementation.

After hind limb suspension, a remodeling of postural muscle phenotype is observed. This remodeling results in a shift of muscle profile from slow-oxidative to fast-glycolytic. These metabolic changes and fiber type shift increase muscle fatigability. Acetyl-L-carnitine (ALCAR) influences the skeletal muscle phenotype of soleus muscle suggesting a positive role of dietary supplementation of ALCAR during unloading. In the present study, we applied a 2-D DIGE, mass spectrometry and biochemical assays, to assess qualitative and quantitative differences in the proteome of rat slow-twitch soleus muscle subjected to disuse. Meanwhile, the effects of ALCAR administration on muscle proteomic profile in both unloading and normal-loading conditions were evaluated. The results indicate a modulation of troponin I and tropomyosin complex to regulate fiber type transition. Associated, or induced, metabolic changes with an increment of glycolytic enzymes and a decreased capacity of fat oxidation are observed. These metabolic changes appear to be counteracted by ALCAR treatment, which restores the mitochondrial mass and decreases the glycolytic enzyme expression, suggesting a normalization of the metabolic shift observed in unloaded animals. This normalization is accompanied by a maintenance of body weight and seems to prevent a switch of fiber type.

A proteomic assessment of muscle contractile alterations during unloading and reloading.

Unloading of skeletal muscle causes atrophy and altered contractility. To identify major muscle proteins responding significantly to the altered loading and to elucidate how the contractile alterations reflect potential proteomic modifications, we examined protein expression in the rat soleus muscle during 3-week hindlimb suspension and 2-week reloading. Compared with unsuspended controls, experimental animals had a 0.5- to 0.6-fold decrease in tension during unloading and early reloading, comparable to 0.2- to 0.6-fold decreases in the protein levels of myosin light chain 1 (MLC1), alpha-actin, tropomyosin beta-chain, and troponins T1 and T2. The observed 1.4- to 1.6-fold increase in shortening velocity appears to reflect 1.2- to 9.0-fold increases in the protein levels of fast-type MLC2, glycolytic enzymes, and creatine kinase, and 0.2- to 0.3-fold decreases in slow-type troponins T1 and T2. The levels of three heat shock proteins (p20, alpha crystallin B chain, and HSP90) decreased during unloading but returned to control levels during reloading. These results imply that proteomic responses to unloading change overall myofibrillar integrity and metabolic regulation, resulting in altered contractility.

Energy expenditure and blood flows in thermoregulatory organs during microgravity simulation in rat. Emphasis on the importance of the control group.

Rat tail suspension is commonly used to mimic human physiology in space. However, energy metabolism adaptation and related autonomic responses are unknown. To give new insights in energy homeostasis, we determined total energy expenditure (TEE) and blood flow redistribution in thermoregulatory organs during suspension using two control groups of animals widely accepted in the literature: the individually housed (isolated) and restraint rats (horizontally attached to the suspension device). Rats (n=33) were randomly assigned during 14-days to three experimental groups: isolated, suspended, or attached. TEE was assessed by a doubly labeled water method throughout the 14 days, and regional blood flow by radiolabeled microsphere procedure at the end of the protocol. Attachment vs. suspension resulted in a significant decrease in TEE (25%), skin (54%), adrenal (55%) and kidney (42%) blood flows, cardiac index (33%), and plasma corticosterone (50%), whereas total peripheral resistances increased (50%). Isolation vs. attachment triggered an inverse response, of similar amplitude, for all above variables. By comparing isolation and suspension, no overall effect was observed. The striking conclusion of this study is that no clear conclusion can be drawn. The choice of the isolated or attached animals as control profoundly influences the outcome results regarding the effects of simulated weightlessness. Further studies are needed but we favor the attached group as the true control since, from a theoretical point of view, a suspended rat is attached plus suspended. In such conditions, TEE decreases to the same extent in rat and humans during simulated microgravity. When reviewing published experiments, we recommend special attention to the control group used rather than on the effects of suspension as compared to an undefined control.

Spatial patterns of atrophied muscle fibers during exercised recovery.

The effect of run training during the recovery period on the spatial distributions of fiber type was examined in atrophic soleus muscle of adult rats following 28 days of hindlimb suspension. During recovery, clusters of damaged and type IIC fibers were observed, which were more pronounced in the exercised animals than in both exercised and nonexercised control groups. The results indicate that exercise during recovery following suspension-induced hindlimb muscle atrophy produces changes in the soleus fiber-type cross-sectional area, both absolute and relative. These changes were not seen in the sedentary recovery group or in control rats exposed to the same exercise regimen. The author concludes that this treatment, unlike neurogenic pathologies, does not cause any remodeling during recovery, in the sense of changed adjacency relations among fiber types.

[Echocardiographic assessment of left ventricular structure and function after simulated weightlessness in rats].

Objective. To investigate whether the changes in rat after simulated weightlessness are similar to those in astronaut after flight. Methods. The effects of 4 wk tail-suspension on left ventricular structure and function in rats were examined by echocardiography. Results. After 4 wk of simulated weightlessness, the thickness of both the anterior and posterior wall in left ventricle (LV) showed a general trend of decrease, but these changes were not statistically significant; the end-systolic and end-diastolic internal dimensions (ESD and EDD respectively) of LV decreased significantly; and the end-systolic volume, end-diastolic volume and stroke volume (ESV, EDV and SV respectively) were all reduced; so did the relevant indices of them. There were no significant differences in ejection fraction (EF) and fractional shortening (FS) between the tail-suspended and control groups. The left ventricular mass (LVM) and its index (LVMI) were decreased. The peak velocities of blood flow of aorta, pulmonary artery and mitral valve didn't show any significant change after simulated weightlessness. Conclusion. Medium-term simulated weightlessness may lead to a significant decrease in left ventricular internal dimension, ventricular volume, and mass, and a trend of decrease in mean left ventricular wall thickness. These changes in rats are similar to those observed in astronauts postflight.

Quantitative assessment of degenerative changes in soleus muscle after hindlimb suspension and recovery.

The aim of this study was to quantify the degenerative and regenerative changes in rat soleus muscle resulting from 3-week hindlimb suspension at 45 degrees tilt (HS group, n = 8) and 4-week normal cage recovery (HS-R group, n = 7). Degenerative changes were quantified by microscope examination of muscle cross sections, and the myosin heavy chain (MHC) composition of soleus muscles was studied by sodium dodecyl sulphate polyacrylamide gel electrophoresis. At the end of 3-week hindlimb suspension, histological signs of muscle degenerative changes were detected in soleus muscles. There was a significant variability in the percentage of fibres referred to as degenerating (%dg) in individual animals in the HS group [%dg = 8.41 (SEM 0.5)%, range 4.66%-14.08%]. Moreover, %dg varied significantly along the length of the soleus muscle. The percentage of fibres with internal nuclei was less than %dg in HS-soleus muscles [4.12 (SEM 0.3)%, range 1.24%-8.86%]. In 4-week recovery rats, the greater part of the fibres that were not referred to as normal, retained central nuclei [15.8 (SEM 2.2)%, range 6.2%-21.1%]. A significant increase in the slow isoform of MHG was recorded in the HS-R rats, compared to muscles from age-matched rats (P < 0.01). These results would suggest that a cycle of myofibre degeneration-regeneration occurred during HS and passive recovery, and that the increased accumulation of slow MHC observed in soleus muscles after recovery from HS could be related to the prevalence of newly formed fibres.