Multi-modal imaging for assessment of tissue-engineered bone in a critical-sized calvarial defect mouse model.

Tissue-engineered bone (TEB) analysis in vivo relies heavily on tissue histological and end-point evaluations requiring the sacrifice of animals at specific time points. Due to differences in animal response to implanted tissues, the conventional analytical methods to evaluate TEB can introduce data inconsistencies. Additionally, the conventional methods increase the number of animals required to provide an acceptable statistical power for hypothesis testing. Alternatively, our non-invasive optical imaging allows for the longitudinal analysis of regenerating tissue, where each animal acts as its own control, thus reducing overall animal numbers. In our 6 month feasibility study, TEB, consisting of a silk protein scaffold with or without differentiated mesenchymal stem cells, was implanted in a critical-sized calvarial defect mouse model. Osteogenesis of the TEB was monitored through signal variation, using magnetic resonance imaging (MRI) and near-infrared (NIR) optical imaging with IRDye® 800CW BoneTagTM (800CW BT, a bone-specific marker used to label osteogenically differentiated mesenchymal stem cells and mineralization). Histological endpoint measurements and computed tomography (CT) were used to confirm imaging findings. Anatomical MRI revealed decreased signal intensity, indicating mineralization, in the TEB compared to the control (i.e. silk scaffold only) at various growth stages. NIR optical imaging results demonstrated a signal intensity increase of the TEB compared to control. Interpretation of the imaging results were confirmed by histological analysis. Specifically, haematoxylin and eosin staining revealing de novo bone in TEB showed that 80% of the defect was covered by TEB, while only 40% was covered for the control. Taken together, these results demonstrate the potential of multi-modal non-invasive imaging to visualize and quantify TEB for the assessment of regenerative medicine strategies. Copyright © 2015 John Wiley & Sons, Ltd.

Upregulated vimentin suggests new areas of neurodegeneration in a model of an alcohol use disorder.

Excessive alcohol intake, characteristic of an alcohol use disorder (AUD), results in neurodegeneration as well as cognitive deficits that may recover in abstinence. Neurodegeneration in psychiatric disorders such as AUDs is due to various effects on tissue integrity. Several groups report that alcohol-induced neurodegeneration and recovery include a role for adult neurogenesis. Therefore, the initial purpose of this study was to investigate the effect of alcohol on the temporal profile of neural progenitor cells using the radial glia marker, vimentin, in a model of an AUD. However, striking vimentin expression throughout corticolimbic regions led, instead, to the discovery of a significant gliosis response in this model. Adult male rats were subjected to a 4-day binge model of an AUD and brains harvested for immunohistochemistry at 0, 2, 4, 7, 14, and 28 days following the last dose of ethanol. A prominent increase in vimentin immunoreactivity was apparent at 4 and 7 days post binge that returned to control levels by 14 days in the corticolimbic regions examined. Vimentin-positive cells co-labeled with glial fibrillary acidic protein (GFAP), which suggested that cells were reactive astrocytes. A second experiment supported that increased vimentin was not primarily due to alcohol withdrawal seizures and is more likely due to alcohol-induced cell death. As this gliosis was remarkably distinct in regions where cell death had not previously been reported in this model, adjacent tissue sections were processed for FluoroJade B staining for cell death. FluoroJade B-positive cells were evident immediately following the last ethanol dose as expected, but were significantly elevated in the hippocampal dentate gyrus and CA3 regions and corticolimbic regions from 2 to 7 days post binge. Intriguingly, vimentin labeling of astrogliosis is more widespread than FluoroJade B labeling of cell death, which suggests that 4-day binge ethanol consumption is more damaging than originally realized.

Similar withdrawal severity in adolescents and adults in a rat model of alcohol dependence.

Alcohol use during adolescence leads to increased risk of developing an alcohol use disorder (AUD) during adulthood. Converging evidence suggests that this period of enhanced vulnerability for developing an AUD may be due to the adolescent's unique sensitivity and response to alcohol. Adolescent rats have been shown to be less sensitive to alcohol intoxication and withdrawal susceptibility; however, age differences in ethanol pharmacokinetics may underlie these effects. Therefore, this study investigated alcohol intoxication behavior and withdrawal severity using a modified Majchrowicz model of alcohol dependence that has been shown to result in similar blood ethanol concentrations (BECs) despite age differences. Adolescent (postnatal day, PND, 35) and adult rats (PND 70+) received ethanol according to this 4-day binge paradigm and were observed for withdrawal behavior for 17h. As expected, adolescents showed decreased sensitivity to alcohol-induced CNS depression as evidenced by significantly lower intoxication scores. Thus, adolescents received significantly more ethanol each day (12.3+/-0.1g/kg/day) than adults (9.2+/-0.2g/kg/day). Despite greater ethanol dosing in adolescent rats, both adolescent and adult groups had comparable peak BECs (344.5+/-10.2 and 338.5+/-7.8mg/dL, respectively). Strikingly, withdrawal severity was similar quantitatively and qualitatively between adolescent and adult rats. Further, this is the first time that withdrawal behavior has been reported for adolescent rats using this model of alcohol dependence. A second experiment confirmed the similarity in BECs at various time points across the binge. These results demonstrate that after consideration of ethanol pharmacokinetics between adults and adolescents by using a model that produces similar BECs, withdrawal severity is nearly identical. This study, in combination with previous reports on ethanol withdrawal in adolescents and adults, suggests only a BEC-dependent effect of ethanol on withdrawal severity regardless of age.