Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for <em>In Vivo</em> Phenotyping and Drug Monitoring

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular ...

Mayo Clinic

Despite the most aggressive medical and surgical treatments, glioblastoma multiforme remains incurable with a median survival of <1 year. We investigated the antitumor potential of a novel viral agent, an attenuated strain of measles virus (MV), derived from the Edmonston vaccine lineage, genetically engineered to produce carcinoembryonic antigen (CEA). CEA production as the virus replicates can serve as a marker of viral gene expression. Infection of a variety of glioblastoma cell lines including U87, U118, and U251 at MOIs 0.1, 1, and 10 resulted in significant cytopathic effect consisting of excessive syncycial formation and massive cell death at 72-96 h from infection. terminal deoxynucleotidyltransferase-mediated nick end labeling assays demonstrated the mechanism of cell death to be predominantly apoptotic. The efficacy of this approach in vivo was examined in BALB/c nude mice by using both s.c. and intracranial orthotopic U87 tumor models. In the s.c. U87 model, mice with established xenografts were treated with a total dose of 8 x 10(7) plaque forming units of MV-CEA, administered i.v. Mice treated with UV light inactivated MV, and untreated mice with established U87 tumors were used as controls. There was statistically significant regression of s.c. tumors (P < 0.001) and prolongation of survival (P = 0.007) in MV-CEA treated animals compared with the two control groups. In the intracranial orthotopic U87 model, there was significant regression of intracranial U87 tumors treated with intratumoral administration of MV-CEA at a total dose of 1.8 x 10(6) plaque forming units as assessed by magnetic resonance image (P = 0.002), and statistically significant prolongation of survival as compared with mice that received UV-inactivated virus and untreated mice (P = 0.02). Histological examination of brains of MV-CEA-treated animals revealed complete regression of the tumor with the presence of a residual glial scar and reactive changes, mainly presence of hemosiderin-laden macrophages. In addition, CEA levels in the peripheral blood in both the s.c. and orthotopic models increased before tumor regression, indicating viral gene expression, and returned to normal when the tumors regressed. Ifnar(ko) CD46 Ge transgenic mice, susceptible to MV infection, were used to assess central nervous system toxicity of MV-CEA. Intracranial administration of MV-CEA into the caudate nucleus of Ifnar(ko) CD46 Ge did not result in clinical neurotoxicity. Pathologic examination demonstrated limited microglial infiltration surrounding the injection site. In summary, MV-CEA has potent antitumor activity against gliomas in vitro, as well as in both s.c. and orthotopic U87 animal models. Monitoring CEA levels in the serum can serve as a low-risk method of detecting viral gene expression during treatment, and could allow dose optimization and individualization of treatment.

Use of a vaccine strain of measles virus genetically engineered to produce carcinoembryonic antigen as a novel therapeutic agent against glioblastoma multiforme.

This study describes investigation of porous photocrosslinked oligo[(polyethylene glycol) fumarate] (OPF) hydrogels as potential matrix for osteoblastic differentiation of marrow stromal cells (MSCs). The porosity and interconnectivity of porous hydrogels were assessed using magnetic resonance microscopy (MRM) as a noninvasive investigative tool that could image the water construct inside the hydrogels at a high-spatial resolution. MSCs were cultured onto the porous hydrogels and cell number was assessed using PicoGreen DNA assay. Our results showed 10% of cells initially attached to the surface of scaffolds. However, cells did not show significant proliferation over a time period of 14 days. MSCs cultured on porous hydrogels had increased alkaline phosphatase activity as well as deposition of calcium, suggesting successful differentiation and maturation to the osteoblastic phenotype. Moreover, continued expression of type I collagen and osteonectin over 14 days confirmed osteoblastic differentiation of MSCs. MRM was also applied to monitor osteogenesis of MSCs on porous hydrogels. MRM images showed porous scaffolds became consolidated with osteogenic progression of cell differentiation. These findings indicate that porous OPF scaffolds enhanced MSC differentiation leading to development of bone-like mineralized tissue.

Effect of hydrogel porosity on marrow stromal cell phenotypic expression.

A controlled study to evaluate a new technique for spinal rod fixation after spinal cord injury in rats. Alignment of implanted tissue-engineered scaffolds was assessed radiographically and by magnetic resonance imaging.

Rigid fixation of the spinal column improves scaffold alignment and prevents scoliosis in the transected rat spinal cord.

Measuring in vivo spinal cord injury and repair remains elusive. Using magnetic resonance spectroscopy (MRS) we examined brainstem N-acetyl-aspartate (NAA) as a surrogate for spinal cord injury in two mouse strains with different reparative phenotypes following virus-induced demyelination. Swiss Jim Lambert (SJL) and Friend Virus B (FVB) mice progressively demyelinate with axonal loss. FVB mice demyelinate similarly but eventually remyelinate coincident with functional recovery. Brainstem NAA levels drop in both but recover in FVB mice. Chronically infected SJL mice lost 30.5% of spinal cord axons compared to FVB mice (7.3%). In remyelination-enhancing or axon-preserving clinical trials, brainstem MRS may be a viable endpoint to represent overall spinal cord dysfunction.

Brainstem 1H nuclear magnetic resonance (NMR) spectroscopy: marker of demyelination and repair in spinal cord.

Genomic perturbations that challenge normal signaling at the pluripotent stage may trigger unforeseen ontogenic aberrancies. Anticipatory systems biology identification of transcriptome landscapes that underlie latent phenotypes would offer molecular diagnosis before the onset of symptoms. The purpose of this study was to assess the impact of calreticulin-deficient embryonic stem cell transcriptomes on molecular functions and physiological systems. Bioinformatic surveillance of calreticulin-null stem cells, a monogenic insult model, diagnosed a disruption in transcriptome dynamics, which re-prioritized essential cellular functions. Calreticulin-calibrated signaling axes were uncovered, and network-wide cartography of undifferentiated stem cell transcripts suggested cardiac manifestations. Calreticulin-deficient stem cell-derived cardiac cells verified disorganized sarcomerogenesis, mitochondrial paucity, and cytoarchitectural aberrations to validate calreticulin-dependent network forecasts. Furthermore, magnetic resonance imaging and histopathology detected a ventricular septal defect, revealing organogenic manifestation of calreticulin deletion. Thus, bioinformatic deciphering of a primordial calreticulin-deficient transcriptome decoded at the pluripotent stem cell stage a reconfigured multifunctional molecular registry to anticipate predifferentiation susceptibility toward abnormal cardiophenotype.

Decoded calreticulin-deficient embryonic stem cell transcriptome resolves latent cardiophenotype.

Acamprosate is clinically used to treat alcoholism. However, the precise molecular functionality of acamprosate in the central nervous system remains unclear, although it is known to antagonize glutamate action in the brain. Since elevated glutamate signaling, especially in the nucleus accumbens (NAc), is implicated in several aspects of alcoholism, we utilized mice lacking type 1 equilibrative nucleoside transporter (ENT1), which exhibit increased glutamate levels in the NAc as well as increased ethanol drinking behaviors. We found that acamprosate significantly reduced ethanol drinking of mice lacking ENT1 (ENT1(-/-)) while having no such effect in wild-type littermates. We then analyzed the basal and acamprosate-treated accumbal metabolite profiles of ENT1(-/-) and wild-type mice using in vivo 16.4T proton magnetic resonance spectroscopy (MRS). Our data show that basal glutamate+glutamine (Glx), glutamate, glutamine and N-acetylaspartatic acid (NAA) levels are increased in the nucleus accumbens (NAc) of ENT1(-/-) compared to wild-type mice. We then found that acamprosate treatment significantly reduced Glx and glutamine levels while increasing taurine levels in the NAc of only ENT1(-/-) compared to their saline-treated group while normalizing other metabolite compared to wild-type mice. This study will be useful in the understanding of the molecular basis of acamprosate in the brain.

Acamprosate reduces ethanol drinking behaviors and alters the metabolite profile in mice lacking ENT1.

Euthanasia: ethical risks.

Acamprosate is clinically used to treat alcohol-dependent patients. While the molecular and pharmacological mechanisms of acamprosate remain unclear, it has been shown to regulate γ-aminobutyric acid (GABA) or glutamate levels in the cortex and striatum. To investigate the effect of acamprosate on brain metabolites in the medial prefrontal cortex (mPFC) and nucleus accumbens (NAc), we employed in vivo 16.4 T proton magnetic resonance spectroscopy. We utilized type 1 equilibrative nucleoside transporter (ENT1) null mice since acamprosate attenuates ethanol drinking in these mice. Our findings demonstrated that ethanol withdrawal reduced GABA levels and increased phosphorylated choline compounds in the mPFC of both wild-type and ENT1 null mice. Notably, acamprosate normalized these withdrawal-induced changes only in ENT1 null mice. In the NAc, ethanol withdrawal increased glutamate and glutamine (Glx) levels only in wild-type mice. Interestingly, acamprosate reduced Glx levels in the NAc compared to the withdrawal state in both genotypes. These results provide a molecular basis for the pharmacological effect of acamprosate in the cortical-striatal circuit.

Ethanol withdrawal-induced brain metabolites and the pharmacological effects of acamprosate in mice lacking ENT1.

The ongoing development of animal models of neurological and psychiatric disorders in combination with the development of advanced nuclear magnetic resonance (NMR) techniques and instrumentation has led to increased use of in vivo proton NMR spectroscopy ((1)H-MRS) for neurochemical analyses. (1)H-MRS is one of only a few analytical methods that can assay in vivo and longitudinal neurochemical changes associated with neurological and psychiatric diseases, with the added advantage of being a technique that can be utilized in both preclinical and clinical studies. In this review, recent progress in the use of (1)H-MRS to investigate animal models of neurological and psychiatric disorders is summarized with examples from the literature and our own work.

Preclinical (1)H-MRS neurochemical profiling in neurological and psychiatric disorders.

The cholangiopathies are a diverse group of biliary tract disorders, many of which lack effective treatment. Murine models are an important tool for studying their pathogenesis, but existing noninvasive methods for assessing biliary disease in vivo are not optimal. Here we report our experience with using micro-computed tomography (microCT) and nuclear magnetic resonance (MR) imaging to develop a technique for live-mouse cholangiography. Using mdr2 knockout (mdr2KO, a model for primary sclerosing cholangitis (PSC)), bile duct-ligated (BDL), and normal mice, we performed in vivo: (1) microCT on a Siemens Inveon PET/CT scanner and (2) MR on a Bruker Avance 16.4 T spectrometer, using Turbo Rapid Acquisition with Relaxation Enhancement, IntraGate Fast Low Angle Shot, and Half-Fourier Acquisition Single-shot Turbo Spin Echo methods. Anesthesia was with 1.5-2.5% isoflurane. Scans were performed with and without contrast agents (iodipamide meglumine (microCT), gadoxetate disodium (MR)). Dissection and liver histology were performed for validation. With microCT, only the gallbladder and extrahepatic bile ducts were visualized despite attempts to optimize timing, route, and dose of contrast. With MR, the gallbladder, extra-, and intrahepatic bile ducts were well-visualized in mdr2KO mice; the cholangiographic appearance was similar to that of PSC (eg, multifocal strictures) and could be improved with contrast administration. In BDL mice, MR revealed cholangiographically distinct progressive dilation of the biliary tree without ductal irregularity. In normal mice, MR allowed visualization of the gallbladder and extrahepatic ducts, but only marginal visualization of the diminutive intrahepatic ducts. One mouse died during microCT and MR imaging, respectively. Both microCT and MR scans could be obtained in ≤20 min. We, therefore, demonstrate that MR cholangiography can be a useful tool for longitudinal studies of the biliary tree in live mice, whereas microCT yields suboptimal duct visualization despite requiring contrast administration. These findings support further development and application of MR cholangiography to the study of mouse models of PSC and other cholangiopathies.

Micro-computed tomography and nuclear magnetic resonance imaging for noninvasive, live-mouse cholangiography.

Archived formalin-fixed paraffin-embedded (FFPE) tissue collections represent a valuable informational resource for numerous studies. However, there is no NMR signal from FFPE specimens at room temperature. To obtain MR images and enable comparison of magnetic resonance microscopy (MRM) and histology studies we propose to image FFPE tissue at elevated temperature.

MR microscopy of formalin fixed paraffin embedded histology specimens.

Noninvasive imaging techniques that quantify renal tissue composition are needed to more accurately ascertain prognosis and monitor disease progression in polycystic kidney disease (PKD). Given the success of magnetization transfer (MT) imaging to characterize various tissue remodeling pathologies, it was tested on a murine model of autosomal dominant PKD.

Prasanna K. Mishra

Department of Biochemistry and Molecular Biology

Mayo Clinic

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

Prasanna K. Mishra

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biochemistry and Molecular Biology

Mayo Clinic

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

Department of Biochemistry and Molecular Biology