Improved Brain Pathology and Progressive Peripheral Neuropathy in a 15 Year Old Survivor of Infantile Krabbe Disease Treated With Umbilical Cord Transplantation.

Objective: Krabbe disease is a fatal leukodystrophy caused by deficiency in galactocerebrosidase enzyme activity. The only currently available therapy is hematopoietic stem cell transplantation with bone marrow or umbilical cord blood (UCBT), which leads to increased lifespan and functional abilities when performed in the preclinical stage. While stabilization of white matter disease has been seen on serial MRI studies, neuropathological changes following transplantation have not been documented so far. Materials and Methods: We report the first postmortem examination of a 15-year-old female patient with infantile Krabbe disease after UCBT in infancy. Results: In contrast to an untreated Krabbe disease brain, which showed severe myelin and oligodendrocyte loss with occasional globoid cells, the transplanted brain displayed markedly improved myelin preservation, but not reaching normal myelination levels. Consistent with the transplanted patient's clinical presentation of pronounced deficits in gross motor skills, corticospinal tracts were most severely affected. No globoid cells or evidence of active demyelination were observed in the central nervous system, indicative of at least partially successful functional restoration. This was corroborated by the identification of male donor-derived cells in the brain by in situ hybridization. Unlike the observed disease stabilization in the central nervous system, the patient experienced progressive peripheral neuropathy. While diminished macrophage infiltration was seen postmortem, peripheral nerves exhibited edema, myelin and axon loss and persistent Schwann cell ultrastructural inclusions. Conclusion: Umbilical cord blood transplantation was able to alter the natural disease progression in the central but less so in the peripheral nervous system, possibly due to limited cross-correction of Schwann cells.

Thioguanosine Conversion Enables mRNA-Lifetime Evaluation by RNA Sequencing Using Double Metabolic Labeling (TUC-seq DUAL).

Temporal information about cellular RNA populations is essential to understand the functional roles of RNA. We have developed the hydrazine/NH4 Cl/OsO4 -based conversion of 6-thioguanosine (6sG) into A', where A' constitutes a 6-hydrazino purine derivative. A' retains the Watson-Crick base-pair mode and is efficiently decoded as adenosine in primer extension assays and in RNA sequencing. Because 6sG is applicable to metabolic labeling of freshly synthesized RNA and because the conversion chemistry is fully compatible with the conversion of the frequently used metabolic label 4-thiouridine (4sU) into C, the combination of both modified nucleosides in dual-labeling setups enables high accuracy measurements of RNA decay. This approach, termed TUC-seq DUAL, uses the two modified nucleosides in subsequent pulses and their simultaneous detection, enabling mRNA-lifetime evaluation with unprecedented precision.

Analyses of Synthetic N-Acyl Dopamine Derivatives Revealing Different Structural Requirements for Their Anti-inflammatory and Transient-Receptor-Potential-Channel-of-the-Vanilloid-Receptor-Subfamily-Subtype-1 (TRPV1)-Activating Properties.

We studied the chemical entities within N-octanoyl dopamine (NOD) responsible for the activation of transient-receptor-potential channels of the vanilloid-receptor subtype 1 (TRPV1) and inhibition of inflammation. The potency of NOD in activating TRPV1 was significantly higher compared with those of variants in which the ortho-dihydroxy groups were acetylated, one of the hydroxy groups was omitted ( N-octanoyl tyramine), or the ester functionality consisted of a bulky fatty acid ( N-pivaloyl dopamine). Shortening of the amide linker (ΔNOD) slightly increased its potency, which was further increased when the carbonyl and amide groups (ΔNODR) were interchanged. With the exception of ΔNOD, the presence of an intact catechol structure was obligatory for the inhibition of VCAM-1 and the induction of HO-1 expression. Because TRPV1 activation and the inhibition of inflammation by N-acyl dopamines require different structural entities, our findings provide a framework for the rational design of TRPV1 agonists with improved anti-inflammatory properties.

Quantitative arterial spin labelling perfusion measurements in rat models of renal transplantation and acute kidney injury at 3T.

OBJECTIVES: To employ ASL for the measurement of renal cortical perfusion in particular renal disorders typically associated with graft loss and to investigate its potential to detect and differentiate the related functional deterioration i.e., in a setting of acute kidney injury (AKI) as well as in renal grafts showing acute and chronic transplant rejection. MATERIALS AND METHODS: 14 Lewis rats with unilateral ischaemic AKI and 43 Lewis rats with renal grafts showing acute or chronic rejections were used. All ASL measurements in this study were performed on a 3T MR scanner using a FAIR True-FISP approach to assess renal blood flow (RBF). Perfusion maps were calculated and the cortical blood flow was determined using a region-of-interest based analysis. RBF of healthy and AKI kidneys as well as of both rejection models, were compared. In a subsample of 20 rats, creatinine clearance was measured and correlated with cortical perfusion. RESULTS: RBF differs significantly between healthy and AKI kidneys (P<0.001) with a mean difference of 213±80ml/100g/min. Renal grafts with chronic rejections show a significantly higher (P<0.001) mean cortical perfusion (346±112ml/100g/min) than grafts with acute rejection (240±66ml/100g/min). Both transplantation models have a significantly (P<0.001) lower perfusion than healthy kidneys. Renal creatinine clearance is significantly correlated (R=0.85, P<0.001) with cortical blood flow. CONCLUSION: Perfusion measurements with ASL have the potential to become a valuable diagnostic tool, regarding the detection of renal impairment and the differentiation of disorders that lead to a loss of renal function and that are typically associated with graft loss.

Radiofluorinated N-Octanoyl Dopamine ([18F]F-NOD) as a Tool To Study Tissue Distribution and Elimination of NOD in Vitro and in Vivo.

To mitigate pretransplantation injury in organs of potential donors, N-octanoyl dopamine (NOD) treatment might be considered as it does not affect hemodynamic parameters in braindead (BD) donors. To better assess optimal NOD concentrations for donor treatment, we report on the fast and facile radiofluorination of the NOD-derivative [18F]F-NOD [18F]5 for in vivo assessment of NOD's elimination kinetics by means of PET imaging. [18F]5 was synthesized in reproducibly high radiochemical yields and purity (>98%) as well as high specific activities (>20 GBq/µmol). Stability tests showed no decomposition of [18F]5 over a period of 120 min in rat plasma. In vitro, low cell association was found for [18F]5, indicating no active transport mechanism into cells. In vivo, [18F]5 exhibited a fast blood clearance and a predominant hepatobiliary elimination. As these data suggest that also NOD might be cleared fast, further pharmacokinetic evaluation is warranted.

N-octanoyl dopamine treatment exerts renoprotective properties in acute kidney injury but not in renal allograft recipients.

BACKGROUND: N-octanoyl dopamine (NOD) treatment improves renal function when applied to brain dead donors and in the setting of warm ischaemia-induced acute kidney injury (AKI). Because it also activates transient receptor potential vanilloid type 1 (TRPV1) channels, we first assessed if NOD conveys its renoprotective properties in warm ischaemia-induced AKI via TRPV1 and secondly, if renal transplant recipients also benefit from NOD treatment. METHODS: We induced warm renal ischaemia in Lewis, wild-type (WT) and TRPV1(-/-) Sprague-Dawley (sd) rats by clamping the left renal artery for 45 min. Transplantations were performed in allogeneic and syngeneic donor-recipient combinations (Fisher to Lewis and Lewis to Lewis) with a cold ischaemia time of 20 h. Treatment was instituted directly after restoration of organ perfusion. Renal function, histology and perfusion were assessed by serum creatinine, microscopy and magnetic resonance imaging (MRI) using arterial spin labelling (ASL). RESULTS: NOD treatment significantly improved renal function in Lewis rats after warm ischaemia-induced AKI. It was, however, not effective after prolonged cold ischaemia. The renoprotective properties of NOD were only observed in Lewis or WT, but not in TRPV1(-/-) sd rats. Renal inflammation was significantly abrogated by NOD. MRI-ASL showed a significantly lower cortical perfusion in ischaemic when compared with non-ischaemic kidneys. No overall differences were observed in renal perfusion between NOD- and NaCl-treated rats. CONCLUSIONS: NOD treatment reduces renal injury in warm ischaemia, but is not effective in renal transplant in our experimental animal models. The salutary effect of NOD appears to be TPRV1-dependent, not involving large changes in renal perfusion.

N-Octanoyl Dopamine for Donor Treatment in a Brain-death Model of Kidney and Heart Transplantation.

BACKGROUND: This study investigated the potential use of N-octanoyl dopamine (NOD) in donor management to ameliorate the damage caused by brain death and ischemia-reperfusion injury in a rat model of kidney and heart transplantation. METHODS: Brain-dead Fisher rats were treated for 6 hours with either saline or saline plus NOD. Orthotopic kidney and heterotopic heart transplantation were performed in different Lewis recipient rats. The right donor kidneys were stored for biochemical analysis. Blood samples were taken from the donor and on several days after transplantation from the recipient. All grafts were harvested after 7 days. RESULTS: There was no effect on donor heart rate and blood pressure under NOD treatment. The release of lactate dehydrogenase (LDH) during brain death was reduced in the NOD group. The right kidneys from NOD-preconditioned animals revealed diminished expression of the proinflammatory cell adhesion molecules intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1). Nevertheless, there was no difference in renal infiltration with ED1 (CD68) or major histocompatibility complex (MHC) class II-positive cells. Recipients receiving a renal allograft from NOD-treated donors had a significantly better renal function at day 1 after transplantation. Banff-grading after 7 days showed significantly reduced scores for tubulitis and vasculitis in the grafts of these recipients. In the heart allograft recipients, lower plasma LDH levels were observed. CONCLUSIONS: Donor preconditioning with NOD leads to better graft function and reduced acute rejection in untreated renal allograft recipients without displaying adverse effects on heart allografts.

Functional imaging of acute kidney injury at 3 Tesla: investigating multiple parameters using DCE-MRI and a two-compartment filtration model.

OBJECT: To investigate how MR-based parameters reflect functional changes in kidneys with acute kidney injury (AKI) using dynamic contrast enhanced MRI and a two-compartment renal filtration model. MATERIALS AND METHODS: MRI data of eight male Lewis rats were analyzed retrospectively. Five animals were subjected to AKI, three native rats served as control. All animals underwent perfusion imaging by dynamic contrast-enhanced MRI. Renal blood volume, glomerular filtration rate (GFR) as well as plasma and tubular mean transit times were estimated from regions-of-interest drawn in the renal cortex. Differences between healthy kidneys and kidneys subjected to AKI were analyzed using a paired t-test. RESULTS: Significant differences between ischemic and healthy kidneys could only be detected for the glomerular filtration rate. For all other calculated parameters, differences were present, however not significant. In rats with AKI, average single kidney GFR was 0.66 ± 0.37 ml/min for contralateral and 0.26 ± 0.12 ml/ min for diseased kidneys (P = 0.0254). For the healthy control group, the average GFR was 0.39 ± 0.06 ml/min and 0.41 ± 0.11 ml/min, respectively. Differences between diseased kidneys of AKI rats and ipsilateral kidneys of the healthy control group were significant (P = 0.0381). CONCLUSION: Significant differences of functional parameters reflecting damage of the renal tissue of kidneys with AKI compared to the contralateral, healthy kidneys could only be detected by GFR. GFR might be a useful parameter that allows for a spatially resolved detection of abnormal changes of renal tissue by AKI.

Renal perfusion in acute kidney injury with DCE-MRI: deconvolution analysis versus two-compartment filtration model.

PURPOSE: To investigate the results of different pharmacokinetic models of a quantitative analysis of renal blood flow (RBF) in acute kidney injury using deconvolution analysis and a two-compartment renal filtration model. MATERIALS AND METHODS: MRI data of ten male Lewis rats were analyzed retrospectively. Six animals were subjected to unilateral acute kidney injury and underwent perfusion imaging by dynamic contrast-enhanced MRI (DCE-MRI). Renal blood flow was estimated from regions-of-interest depicting the cortex in the DCE-MRI perfusion maps. The perfusion models were compared by a paired t-test and Bland-Altman plots. RESULTS: No significant difference was found between the two compartment model and the deconvolution analysis (P=0.2807). Differences between healthy and diseased kidney in the AKI model were significant for both methods (P<0.05). A Bland-Altman plot showed no systematic errors, and values were equally distributed around the mean difference between the methods lying within the range of 1.96 standard deviations. CONCLUSION: Both quantification strategies could detect the kidneys that were impaired by AKI. When just aiming at RBF as a marker, a deconvolution analysis can provide similar values as the 2CFM. If functional parameters beyond RBF like glomerular filtration rate are needed, the 2CFM should be employed.

Quantitative renal perfusion measurements in a rat model of acute kidney injury at 3T: testing inter- and intramethodical significance of ASL and DCE-MRI.

OBJECTIVES: To establish arterial spin labelling (ASL) for quantitative renal perfusion measurements in a rat model at 3 Tesla and to test the diagnostic significance of ASL and dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) in a model of acute kidney injury (AKI). MATERIAL AND METHODS: ASL and DCE-MRI were consecutively employed on six Lewis rats, five of which had a unilateral ischaemic AKI. All measurements in this study were performed on a 3 Tesla MR scanner using a FAIR True-FISP approach and a TWIST sequence for ASL and DCE-MRI, respectively. Perfusion maps were calculated for both methods and the cortical perfusion of healthy and diseased kidneys was inter- and intramethodically compared using a region-of-interest based analysis. RESULTS/SIGNIFICANCE: Both methods produce significantly different values for the healthy and the diseased kidneys (P<0.01). The mean difference was 147±47 ml/100 g/min and 141±46 ml/100 g/min for ASL and DCE-MRI, respectively. ASL measurements yielded a mean cortical perfusion of 416±124 ml/100 g/min for the healthy and 316±102 ml/100 g/min for the diseased kidneys. The DCE-MRI values were systematically higher and the mean cortical renal blood flow (RBF) was found to be 542±85 ml/100 g/min (healthy) and 407±119 ml/100 g/min (AKI). CONCLUSION: Both methods are equally able to detect abnormal perfusion in diseased (AKI) kidneys. This shows that ASL is a capable alternative to DCE-MRI regarding the detection of abnormal renal blood flow. Regarding absolute perfusion values, nontrivial differences and variations remain when comparing the two methods.