The metabolic status of the kidney is a determinant of injury susceptibility and a measure of progression for many disease processes; however, noninvasive modalities to assess kidney metabolism are ...lacking. In this study, we employed positron emission tomography (PET) and intravital multiphoton microscopy (MPM) to assess cortical and proximal tubule glucose tracer uptake, respectively, following experimental perturbations of kidney metabolism. Applying dynamic image acquisition PET with 2-
fluoro-2-deoxyglucose (
F-FDG) and tracer kinetic modeling, we found that an intracellular compartment in the cortex of the kidney could be distinguished from the blood and urine compartments in animals. Given emerging literature that the tumor suppressor protein p53 is an important regulator of cellular metabolism, we demonstrated that PET imaging was able to discern a threefold increase in cortical
F-FDG uptake following the pharmacological inhibition of p53 in animals. Intravital MPM with the fluorescent glucose analog 2-
-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino-2-deoxyglucose (2-NBDG) provided increased resolution and corroborated these findings at the level of the proximal tubule. Extending our observation of p53 inhibition on proximal tubule glucose tracer uptake, we demonstrated by intravital MPM that pharmacological inhibition of p53 diminishes mitochondrial potential difference. We provide additional evidence that inhibition of p53 alters key metabolic enzymes regulating glycolysis and increases intermediates of glycolysis. In summary, we provide evidence that PET is a valuable tool for examining kidney metabolism in preclinical and clinical studies, intravital MPM is a powerful adjunct to PET in preclinical studies of metabolism, and p53 inhibition alters basal kidney metabolism.
A growing number of studies have investigated combination treatment as an approach to treat bone disease. The goal of this study was to investigate the combination of alendronate and raloxifene with ...a particular focus on mechanical properties. To achieve this goal we utilized a large animal model, the beagle dog, used previously by our laboratory to study both alendronate and raloxifene monotherapies. Forty-eight skeletally mature female beagles (1-2 years old) received daily oral treatment: saline vehicle (VEH), alendronate (ALN), raloxifene (RAL) or both ALN and RAL. After 6 and 12 months of treatment, all animals underwent assessment of bone material properties using in vivo reference point indentation (RPI) and skeletal hydration using ultra-short echo magnetic resonance imaging (UTE-MRI). End point measures include imaging, histomorphometry, and mechanical properties. Bone formation rate was significantly lower in iliac crest trabecular bone of animals treated with ALN (-71%) and ALN+RAL (-81%) compared to VEH. In vivo assessment of properties by RPI yielded minimal differences between groups while UTE-MRI showed a RAL and RAL+ALN treatment regimens resulted in significantly higher bound water compared to VEH (+23 and +18%, respectively). There was no significant difference among groups for DXA- or CT-based measures lumbar vertebra, or femoral diaphysis. Ribs of RAL-treated animals were smaller and less dense compared to VEH and although mechanical properties were lower the material-level properties were equivalent to normal. In conclusion, we present a suite of data in a beagle dog model treated for one year with clinically-relevant doses of alendronate and raloxifene monotherapies or combination treatment with both agents. Despite the expected effects on bone remodeling, our study did not find the expected benefit of ALN to BMD or structural mechanical properties, and thus the viability of the combination therapy remains unclear.
Background:
Alveolar bone grafting is utilized to manage alveolar clefts in patients with cleft lip and palate. However, the timing of bone grafting is variable with conflicting evidence supporting ...the use of primary alveolar bone grafting (PABG) in clinical practice.
Primary Aim:
To provide a qualitative systematic review analysis of long-term outcomes after PABG.
Materials and Methods:
A qualitative systematic review was performed following the Cochrane Handbook and reported using Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. Summative findings were evaluated using Confidence in the Evidence from Reviews of Qualitative research to assess the quality of evidence supporting the findings.
Results:
After removing duplication, 2182 publications were identified, and 2131 were excluded after screening through titles and abstracts. Inclusion criteria for this study included patients who underwent PABG at 24 months of age or younger and a minimum of 5 year follow-up. Thirty-two publications met the inclusion criteria and were included for qualitative analysis. Primary outcome measures included cephalometric analysis, bone graft survival, occlusal analysis, hypomineralization, tooth eruption, radiograph analysis, and arch relationships. Four assessment themes were characterized from the systematic review: (1) bone graft survival, (2) craniofacial skeletal relationships, (3) occlusion and arch forms, and (4) recommendations for utilizing PABG in practice.
Conclusion:
The reported systematic review provides evidence that performing PABG leads to poor long-term outcomes related to bone graft survival and maxillary growth restriction despite some reported positive outcomes.
Transmembrane protein 67 (TMEM67) is mutated in Meckel Gruber Syndrome type 3 (MKS3) resulting in a pleiotropic phenotype with hydrocephalus and renal cystic disease in both humans and rodent models. ...The precise pathogenic mechanisms remain undetermined. Herein it is reported for the first time that a point mutation of TMEM67 leads to a gene dose-dependent hydrocephalic phenotype in the Wistar polycystic kidney (Wpk) rat. Animals with TMEM67 heterozygous mutations manifest slowly progressing hydrocephalus, observed during the postnatal period and continuing into adulthood. These animals have no overt renal phenotype. The TMEM67 homozygous mutant rats have severe ventriculomegaly as well as severe polycystic kidney disease and die during the neonatal period. Protein localization in choroid plexus epithelial cells indicates that aquaporin 1 and claudin-1 both remain normally polarized in all genotypes. The choroid plexus epithelial cells may have selectively enhanced permeability as evidenced by increased Na
, K
and Cl
in the cerebrospinal fluid of the severely hydrocephalic animals. Collectively, these results suggest that TMEM67 is required for the regulation of choroid plexus epithelial cell fluid and electrolyte homeostasis. The Wpk rat model, orthologous to human MKS3, provides a unique platform to study the development of both severe and mild hydrocephalus.
Traumatic brain injury (TBI) results in systemic inflammatory responses that affect the lung. This is especially critical in the setting of lung transplantation, where more than half of donor ...allografts are obtained postmortem from individuals with TBI. The mechanism by which TBI causes pulmonary dysfunction remains unclear but may involve the interaction of high-mobility group box-1 (HMGB1) protein with the receptor for advanced glycation end products (RAGE). To investigate the role of HMGB1 and RAGE in TBI-induced lung dysfunction, RAGE-sufficient (wild-type) or RAGE-deficient (RAGE(-/-)) C57BL/6 mice were subjected to TBI through controlled cortical impact and studied for cardiopulmonary injury. Compared to control animals, TBI induced systemic hypoxia, acute lung injury, pulmonary neutrophilia, and decreased compliance (a measure of the lungs' ability to expand), all of which were attenuated in RAGE(-/-) mice. Neutralizing systemic HMGB1 induced by TBI reversed hypoxia and improved lung compliance. Compared to wild-type donors, lungs from RAGE(-/-) TBI donors did not develop acute lung injury after transplantation. In a study of clinical transplantation, elevated systemic HMGB1 in donors correlated with impaired systemic oxygenation of the donor lung before transplantation and predicted impaired oxygenation after transplantation. These data suggest that the HMGB1-RAGE axis plays a role in the mechanism by which TBI induces lung dysfunction and that targeting this pathway before transplant may improve recipient outcomes after lung transplantation.
The purinergic receptor subtype 7 (P2X7R) represents a novel molecular target for imaging neuroinflammation via PET. GSK1482160, a potent P2X7R antagonist, has high receptor affinity, high ...blood-brain barrier penetration, and the ability to be radiolabeled with
C. We report the initial physical and biologic characterization of this novel ligand.
C-GSK1482160 was synthesized according to published methods. Cell density studies were performed on human embryonic kidney cell lines expressing human P2X7R (HEK293-hP2X7R) and underwent Western blotting, an immunofluorescence assay, and radioimmunohistochemistry analysis using P2X7R polyclonal antibodies. Receptor density and binding potential were determined by saturation and association-disassociation kinetics, respectively. Peak immune response to lipopolysaccharide treatment in mice was determined in time course studies and analyzed via Iba1 and P2X7R Western blotting and Iba1 immunohistochemistry. Whole-animal biodistribution studies were performed on saline- or lipopolysaccharide-treated mice at 15, 30, and 60 min after radiotracer administration. Dynamic in vivo PET/CT was performed on the mice at 72 h after administration of saline, lipopolysaccharide, or lipopolysaccharide + blocking, and 2-compartment, 5-parameter tracer kinetic modeling of brain regions was performed.
P2X7R changed linearly with concentrations or cell numbers. For high-specific-activity
C-GSK1482160, receptor density and K
were 1.15 ± 0.12 nM and 3.03 ± 0.10 pmol/mg, respectively, in HEK293-hP2X7R membranes. Association constant
, dissociation constant
, and binding potential (
/
) in HEK293-hP2X7R cells were 0.2312 ± 0.01542 min
⋅nM
, 0.2547 ± 0.0155 min
, and 1.0277 ± 0.207, respectively. Whole-brain Iba1 expression in lipopolysaccharide-treated mice peaked by 72 h on immunohistochemistry, and Western blot analysis of P2X7R for saline- and lipopolysaccharide-treated brain sections showed a respective 1.8- and 1.7-fold increase in signal enhancement at 72 h. Biodistribution of
C-GSK1482160 in saline- and lipopolysaccharide-treated mice at 72 h was statistically significant across all tissues studied. In vivo dynamic
C-GSK1482160 PET/CT of mice at 72 h after administration of saline, lipopolysaccharide, or lipopolysaccharide + blocking showed a 3.2-fold increase and 97% blocking by 30 min. The total distribution volumes for multiple cortical regions and the hippocampus showed statistically significant increases and were blocked by an excess of authentic standard GSK1482160.
The current study provides compelling data that support the suitability of
C-GSK1482160 as a radioligand targeting P2X7R, a biomarker of neuroinflammation.
Recent advancements in PET instrumentation have made the non-invasive assessment of cardiovascular function in small animals a reality. The majority of small animal PET systems use stationary ...detector gantries, thus affording high temporal resolution imaging of cardiac function. Systems designed to maximize spatial resolution and detection sensitivity employing rotating gantry designs are suboptimal when high temporal resolution imaging is needed. To overcome this limitation, the current work developed a novel view-sharing data analysis scheme suitable for dynamic cardiac PET imaging using
18
F-NaF as the tracer and tracer kinetic model analysis. This scheme was tested in a rat model of cardiovascular function where the relationship between direct transonic flow measures of cardiac output were highly correlated (
f
(
x
) = 1.0216
x
− 24.233,
R
= 0.9158,
p
< 0.001) with the new model. Similarly, derived measures of stroke volume were also highly correlated (
f
(
x
) = 0.9655
x
− 0.0428,
R
= 0.9453,
p
< 0.001) with the current approach. Administration of xylazine caused a statistically significant increase in stroke volume (0.32 ± 0.07 ml,
p
= 0.003,
n
= 4) and a significant decrease in both heart rate (−155 ± 7.1 beats/min,
p
< 0.001,
n
= 4) and cardiac output (−75.9 ± 23.0 ml/kg min,
p
= 0.01,
n
= 4). These findings suggest that the new sinogram binning and kinetic modeling methods produce reliable cardiac function measures suitable for longitudinal monitoring of cardiovascular function.
The purpose of the present study was to develop and validate noninvasive bioluminescence imaging methods for differentially monitoring primary and abdominal metastatic tumor growth in mouse ...orthotopic models of pancreatic cancer.
A semiautomated maximum entropy segmentation method was implemented for the primary tumor region of interest, and a rule-based method for manually drawing a region of interest for the abdominal metastatic region was developed for monitoring tumor growth in orthotopic models of pancreatic cancer. The 2 region-of-interest methods were validated by having 2 observers independently segment Panc-1 tumors, and the results were compared with the number of mesenteric lymph node nodules and histopathologic assessment of liver metastases. The findings were extended to orthotopic tumors of the more metastatic MIA PaCa-2 and AsPC-1 cells where separate groups of animals were implanted with different numbers of cells.
The results demonstrated that the segmentation methods were highly reliable, reproducible, and robust and allowed statistically significant discrimination in the growth rates of primary and abdominal metastatic tumors of different cell lines implanted with different numbers of cells.
The present results demonstrate that primary tumors and abdominal metastatic foci in orthotopic pancreatic cancer models can be reliably quantified separately and noninvasively over time with bioluminescence imaging.
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