Histology and Gadolinium Distribution in the Rodent Brain After the Administration of Cumulative High Doses of Linear and Macrocyclic Gadolinium-Based Contrast Agents.

OBJECTIVES: Retrospective studies in patients with primary brain tumors or other central nervous system pathologies as well as postmortem studies have suggested that gadolinium (Gd) deposition occurs in the dentate nucleus (DN) and globus pallidus (GP) after multiple administrations of primarily linear Gd-based contrast agents (GBCAs). However, this deposition has not been associated with any adverse effects or histopathological alterations. The aim of this preclinical study was to systematically examine differences between linear and macrocyclic GBCAs in their potential to induce changes in brain and skin histology including Gd distribution in high spatial resolution. MATERIALS AND METHODS: Fifty male Wistar-Han rats were randomly allocated into control (saline, n = 10 rats) and 4 GBCA groups (linear GBCAs: gadodiamide and gadopentetate dimeglumine, macrocyclic GBCAs: gadobutrol and gadoteridol; n = 10 rats per group). The animals received 20 daily intravenous injections at a dose of 2.5 mmol Gd/kg body weight. Eight weeks after the last GBCA administration, the animals were killed, and the brain and skin samples were histopathologically assessed (hematoxylin and eosin; cresyl violet [Nissl]) and by immunohistochemistry. The Gd concentration in the skin, bone, brain, and skeletal muscle samples were analyzed using inductively coupled plasma mass spectroscopy (ICP-MS, n = 4). The spatial Gd distribution in the brain and skin samples was analyzed in cryosections using laser ablation coupled with ICP-MS (LA-ICP-MS, n = 3). For the ultra-high resolution of Gd distribution, brain sections of rats injected with gadodiamide or saline (n = 1) were assessed by scanning electron microscopy coupled to energy dispersive x-ray spectroscopy and transmission electron microscopy, respectively. RESULTS: No histological changes were observed in the brain. In contrast, 4 of 10 animals in the gadodiamide group but none of the animals in other groups showed macroscopic and histological nephrogenic systemic fibrosis-like skin lesions. The Gd concentrations observed in the skin/brain samples (in nanomole Gd per gram of tissue) for each agent were as follows: gadodiamide: 1472 ± 115/11.1 ± 5.1, gadopentetate dimeglumine: 80.8 ± 6.2/13.1 ± 7.3, gadobutrol: 1.1 ± 0.5/0.7 ± 0.4, and gadoteridol: 1.7 ± 0.8/0.5 ± 0.2. The average detected residual Gd concentration in the brain was approximately 15-fold higher for linear than for macrocyclic GBCAs. The highest amounts of Gd found in brain corresponded to less than 0.0002% of the injected dose per gram of tissue. Using LA-ICP-MS, high Gd concentrations in the deep cerebellar nuclei and in the granular layer of the cerebellar cortex were detected only for linear gadodiamide and gadopentetate dimeglumine but not for gadoteridol or gadobutrol. The energy dispersive x-ray spectroscopy analysis revealed Gd-containing spots in the skin of animals administered gadodiamide and gadopentetate dimeglumine. Transmission electron microscopy revealed several Gd-containing spots in the region of the dentate nuclei in the brain of 1 animal injected with gadodiamide. CONCLUSIONS: After repeated high dosing, nephrogenic systemic fibrosis-like macroscopic and histopathological lesions of the skin were observed only in some of the gadodiamide-treated animals. No histopathological findings were detected in the rodent brain. The administration of linear GBCAs was associated with significantly higher Gd concentrations in the brain and skin compared with macrocyclic GBCA administration. The results of LA-ICP-MS demonstrated local accumulation of Gd within the deep cerebellar nuclei and the granular layer only after the administration of linear agents. In summary, the detected low Gd concentrations in the skin and brain were well correlated with the higher kinetic stability of macrocyclic GBCA.

Penetration and distribution of gadolinium-based contrast agents into the cerebrospinal fluid in healthy rats: a potential pathway of entry into the brain tissue.

OBJECTIVE: Signal hyperintensity on unenhanced MRI in certain brain regions has been reported after multiple administrations of some, but not all, gadolinium-based contrast agents (GBCAs). One potential initial pathway of GBCA entry into the brain, infiltration from blood into the cerebrospinal fluid (CSF), was systematically evaluated in this preclinical study. METHODS: GBCA infiltration and distribution in the CSF were investigated in healthy rats using repeated fluid-attenuated MRI up to 4 h after high-dose (1.8 mmol/kg) administration of six marketed and one experimental GBCA. Additionally, gadolinium measurements in CSF, blood and brain tissue samples (after 24 h) were performed using inductively coupled plasma mass spectrometry. RESULTS: Enhanced MRI signals in the CSF spaces with similar distribution kinetics were observed for all GBCAs. No substantial differences in the gadolinium concentrations among the marketed GBCAs were found in the CSF, blood or brain tissue. After 4.5 h, the concentration in the CSF was clearly higher than in blood but was almost completely cleared and lower than the brain tissue concentration after 24 h. CONCLUSIONS: In contrast to the brain signal hyperintensities, no differences in penetration and distribution into the CSF of healthy rats exist among the marketed GBCAs. KEY POINTS: • Gadolinium-based contrast agents can cross the blood-CSF barrier. • Fluid-attenuated MRI shows GBCA distribution with CSF flow. • GBCA structure and physicochemical properties do not impact CSF penetration and distribution. • GBCA clearance from CSF was almost complete within 24 h in rats. • CSF is a potential pathway of GBCA entry into the brain.

Signal Increase on Unenhanced T1-Weighted Images in the Rat Brain After Repeated, Extended Doses of Gadolinium-Based Contrast Agents: Comparison of Linear and Macrocyclic Agents.

OBJECTIVES: In this prospective preclinical study, we evaluated T1-weighted signal intensity in the deep cerebellar nuclei (CN) and globus pallidus (GP) up to 24 days after repeated administration of linear and macrocyclic gadolinium-based contrast agents (GBCAs) using homologous imaging and evaluation methods as in the recently published retrospective clinical studies. In a second part of the study, cerebrospinal fluid (CSF) spaces were evaluated for contrast enhancement by fluid-attenuated magnetic resonance imaging (MRI). MATERIALS AND METHODS: Sixty adult male Wistar-Han rats were randomly divided into a control and 5 GBCA groups (n = 10 per group). The administered GBCAs were gadodiamide, gadopentetate dimeglumine, and gadobenate dimeglumine (linear GBCAs) as well as gadobutrol and gadoterate meglumine (macrocyclic GBCAs) and saline (control). Over a period of 2 weeks, the animals received 10 intravenous injections at a dose of 2.5 mmol Gd/kg body weight, each on 5 consecutive days per week. Before GBCA administration, as well as 3 and 24 days after the last injection, a whole-brain MRI was performed using a standard T1-weighted 3-dimensional turbo spin echo sequence on a clinical 1.5 T scanner. The ratios of signal intensities in deep CN to pons (CN/Po) and GP to thalamus (GP/Th) were determined. For the evaluation of the CSF spaces, 18 additional rats were randomly divided into 6 groups (n = 3 per group) that received the same GBCAs as in the first part of the study. After MR cisternography for anatomical reference, a fluid-attenuated inversion recovery sequence was performed before and 1 minute after intravenous injection of a dose of 1 mmol Gd/kg body weight GBCA or saline. RESULTS: A significantly increased signal intensity ratio of CN/Po was observed 3 and 24 days after the last injection of gadodiamide and gadobenate dimeglumine. No significant changes were observed between the 2 time points. Gadopentetate dimeglumine injection led to a moderately elevated but statistically not significant CN/Po signal intensity ratio. No increased CN/Po signal intensity ratios were determined in the MRI scans of rats that received macrocyclic GBCAs gadobutrol and gadoterate meglumine or saline. The ratio of signal intensity in GP/Th was not elevated in any group injected with GBCAs or saline. Enhanced signal intensities of CSF spaces were observed in the postcontrast fluid-attenuated inversion recovery images of all animals receiving GBCAs but not for saline. CONCLUSIONS: In this animal study in rats, increased signal intensity in the CN was found up to 24 days after multiple, extended doses of linear GBCAs. However, in contrast to clinical reports, the signal enhancement in the GP was not reproduced, demonstrating the limitations of this animal experiment. The elevated signal intensities remained persistent over the entire observation period. In contrast, no changes of signal intensities in either the CN or the GP were observed for macrocyclic GBCAs. However, all GBCAs investigated were able to pass the blood-CSF barrier in rats to a certain, not yet quantified extent.

Divergent and conserved roles of Dll1 signaling in development of craniofacial and trunk muscle.

Craniofacial and trunk skeletal muscles are evolutionarily distinct and derive from cranial and somitic mesoderm, respectively. Different regulatory hierarchies act upstream of myogenic regulatory factors in cranial and somitic mesoderm, but the same core regulatory network - MyoD, Myf5 and Mrf4 - executes the myogenic differentiation program. Notch signaling controls self-renewal of myogenic progenitors as well as satellite cell homing during formation of trunk muscle, but its role in craniofacial muscles has been little investigated. We show here that the pool of myogenic progenitor cells in craniofacial muscle of Dll1(LacZ/Ki) mutant mice is depleted in early fetal development, which is accompanied by a major deficit in muscle growth. At the expense of progenitor cells, supernumerary differentiating myoblasts appear transiently and these express MyoD. The progenitor pool in craniofacial muscle of Dll1(LacZ/Ki) mutants is largely rescued by an additional mutation of MyoD. We conclude from this that Notch exerts its decisive role in craniofacial myogenesis by repression of MyoD. This function is similar to the one previously observed in trunk myogenesis, and is thus conserved in cranial and trunk muscle. However, in cranial mesoderm-derived progenitors, Notch signaling is not required for Pax7 expression and impinges little on the homing of satellite cells. Thus, Dll1 functions in satellite cell homing and Pax7 expression diverge in cranial- and somite-derived muscle.

Adverse skin reactions to iodinated x-ray contrast agents in healthy rats.

PURPOSE: The aim of this preclinical study on healthy Sprague-Dawley rats was to determine whether differences exist in the induction of adverse skin reactions after the intravenous administration of a monomeric and 2 dimeric iodinated nonionic contrast agents. MATERIALS AND METHODS: After intravenous injection of iopromide (monomeric), iodixanol (dimeric), and iotrolan (dimeric) at a dose of 4 g iodine/kg of body weight, mechanical ear volume measurements (10 minute after injection) and intravital microscopy (baseline, 5 minutes after injection) of the ear with the near-infrared dye indocyanine green were performed to determine the volume change and plasma extravasation. Histopathological analysis (20 minutes, 1 hour, and 3 hours after injection) was performed to diagnose alterations in the skin. Blood plasma was analyzed to identify elevated levels of histamine (5 minutes after injection) and inflammatory markers (a multianalyte profile of 58 markers; 1 hour and 3 hours after injection). RESULTS: Only iodixanol induced immediate angioedema formation, with a 100% incidence rate and with slight mast cell infiltration in the ear, muzzle, and paws. The ear showed a 53% volume increase and strong extravasation of plasma proteins into the interstitium, which correlated with highly (11-fold) increased plasma histamine levels 5 minutes after injection. Elevated levels of tumor necrosis factor-α (7.1-fold), macrophage inflammatory protein (MIP)-1α (3.2-fold), and MIP-2 (7.7-fold) were identified 1 hour after the iodixanol injection. Increased levels (fold-change) of MIP-1ß (14; 6.3), monocyte chemotactic protein-1 (3.3; 3.7), monocyte chemotactic protein-3 (2.4; 3.0), stem cell factor (1.7; 2), vascular endothelial growth factor (2; 2.1), and interferon gamma-induced protein-10 (4.1; 39.1) were identified 1 hour and 3 hours after the iodixanol administration, respectively. The level of these molecules remained unchanged after the iopromide and iotrolan injections (except for stem cell factor). CONCLUSIONS: A reversible anaphylactoid-like reaction in healthy Sprague-Dawley rats was observed after the iodixanol administration but not after the monomeric iopromide or dimeric iotrolan injections. Therefore, we conclude that the induction of adverse skin reactions is not per se because of a class effect of dimeric contrast agent.

Contrast media viscosity versus osmolality in kidney injury: lessons from animal studies.

Iodinated contrast media (CM) can induce acute kidney injury (AKI). CM share common iodine-related cytotoxic features but differ considerably with regard to osmolality and viscosity. Meta-analyses of clinical trials generally failed to reveal renal safety differences of modern CM with regard to these physicochemical properties. While most trials' reliance on serum creatinine as outcome measure contributes to this lack of clinical evidence, it largely relies on the nature of prospective clinical trials: effective prophylaxis by ample hydration must be employed. In everyday life, patients are often not well hydrated; here we lack clinical data. However, preclinical studies that directly measured glomerular filtration rate, intrarenal perfusion and oxygenation, and various markers of AKI have shown that the viscosity of CM is of vast importance. In the renal tubules, CM become enriched, as water is reabsorbed, but CM are not. In consequence, tubular fluid viscosity increases exponentially. This hinders glomerular filtration and tubular flow and, thereby, prolongs intrarenal retention of cytotoxic CM. Renal cells become injured, which triggers hypoperfusion and hypoxia, finally leading to AKI. Comparisons between modern CM reveal that moderately elevated osmolality has a renoprotective effect, in particular, in the dehydrated state, because it prevents excessive tubular fluid viscosity.

The effect of iodinated contrast agent properties on renal kinetics and oxygenation.

OBJECTIVE: We analyzed renal kinetics and renal oxygenation in rats after administration of several classes and formulations of contrast agents (CAs) with a focus on the influence of osmolality and substance-specific properties. MATERIALS AND METHODS: We investigated the renal kinetics of a nonionic, dimeric CA (iodixanol) formulated in 3 different osmolalities (hypo-osmolar, iso-osmolar, low-osmolar) and compared it to nonionic, low-osmolar (iopromide), and ionic, low-osmolar CAs (ioxaglate) using computed tomography for a period of 24 hours. The CAs were administered intravenously at a dosage of 4 g iodine/kg body weight. The average exposure was calculated, and urine viscosities were compared before the injection and during the time intervals of 0 to 60 minutes and 60 to 120 minutes after the injection. Renal oxygenation levels of the renal cortex and medulla were estimated using blood-oxygen-level-dependent magnetic resonance imaging. We used histologic methods to systematically analyze the gravity of vacuole formation based on the physicochemical and substance-specific properties of each CA. RESULTS: Iso-osmolar and hypo-osmolar iodixanol and, to a lesser extent, iodixanol/mannitol accumulated rapidly in the kidneys during the first 5 minutes of the injection and remained higher 2, 4, 6, and 24 hours after the injection compared with iopromide and ioxaglate, which showed fast iodine excretion. Similarly, lower renal blood oxygen levels were estimated for all iodixanol formulations as compared with ioxaglate and iopromide. The incidence of vacuole formation was high for all iodixanol formulations and for ioxaglate (6 of 6 rats) and low for iopromide (1 of 6 rats). Moderate severity of vacuoles was determined for the iodixanol solutions; minimal severity, for ioxaglate and iopromide. CONCLUSIONS: We identified a superior profile for the low-osmolar CAs compared with the iso-osmolar CAs regarding rapid excretion, short-term renal exposure, and renal oxygenation.

The osmolality of nonionic, iodinated contrast agents as an important factor for renal safety.

OBJECTIVE: Nonionic iodinated contrast agents (CAs) can be divided into monomeric, low-osmolar, and dimeric, iso-osmolar classes. In clinical practice, renal tolerance of CAs is a concern, especially in patients with impaired renal function. With regard to renal safety, we wanted to evaluate the role of osmolality and viscosity in renal tolerance. MATERIAL AND METHODS: We generated a formulation (iodixanol/mannitol) consisting of the dimeric iodixanol with an osmolality of the monomeric iopromide. Male Han-Wistar rats were intravenously injected with low-osmolar iopromide 300, iso-osmolar iodixanol 320, and iodixanol/mannitol. Saline and diatrizoate were used as controls. A total number of 227 rats were used in the following experiments. We compared the impact of osmolality on renal iodine retention using computed tomography 2 and 24 hours postinjection (p.i.). The animals were killed 2, 24, and 72 hours after injection, and the kidneys were excised for further investigations. Changes in renal cell proliferation were analyzed by 5-bromo-2'-deoxyuridine incorporation 48 hours p.i. as a degree of tissue regeneration after induced injury. To specify potential renal injury, we quantified the expression of acute kidney injury (AKI) markers (kidney injury marker-1 [KIM-1], neutrophil gelatinase-associated lipocalin [NGAL], and plasminogen activator inhibitor-1 [PAI-1]) by quantitative real-time polymerase chain reaction. Furthermore, the kidneys were analyzed histologically, including immunofluorescence analysis. RESULTS: After intravenous application of the CAs into Han-Wistar rats, renal iodine concentration was increased (3-fold) for iodixanol 2 hours p.i. and iodine retention was detected to be prolonged 24 hours p.i. compared with iopromide injection (iodixanol, 520 ± 50 Hounsfield Units [HU] vs iopromide, 42 ± 5 HU). The higher iodine concentration 2 hours p.i. upon iodixanol injection was reduced almost to the level of iopromide when injecting iodixanol/mannitol (iopromide: 289 ± 68 HU vs iodixanol/mannitol: 343 ± 68 HU). In addition, iodixanol application induced increased renal cell proliferation (2.7-fold vs saline), indicating renal injury, which was significantly lower in iopromide-treated animals (1.6-fold vs saline). More detailed analysis of markers for AKI revealed that iodixanol significantly induced the expression of PAI-1 (7.7-fold at 2 hours) as well as KIM-1 (2.1-fold) and NGAL (3.2-fold) at 2 and 24 hours when compared with saline treatment. In contrast, the expression of markers for AKI was low after iopromide (1.4-fold NGAL, 1.7-fold PAI-1, KIM-1 not significant) and iodixanol/mannitol (1.6-fold NGAL, 2.6-fold PAI-1, KIM-1 not significant) injection. CONCLUSION: The present results clearly show that prolonged iodine retention and the enhanced expression of kidney injury markers are caused mainly by the explicitly higher urine viscosity induced by iodixanol. We conclude that the osmolality of low-osmolar CAs such as iopromide induces a positive diuretic effect that is responsible for rapid iodine clearance and prevents increased expression of acute injury markers in the kidney.

Changes of renal water diffusion coefficient after application of iodinated contrast agents: effect of viscosity.

OBJECTIVE: X-ray contrast agents (CA) possess specific physicochemical properties and are excreted renally by glomerular filtration. Thereby, they may affect the diffusion of water molecules within the kidney. The aim of our preclinical study was to investigate potential changes in the apparent diffusion coefficient (ADC) of the kidney after administration of monomeric, low-osmolar, and dimeric, iso-osmolar CA by using diffusion-weighted magnetic resonance imaging (DWI). MATERIAL AND METHODS: First, the relationship between CA viscosity and the ADC of water was assessed by phantom measurements. Subsequently, Han Wistar rats (8 per group) received an intravenous injection of iso-osmolar CA (iodixanol) or low-osmolar CA (iopromide) at a dosage of 4 gI/kg body weight. The control group received saline (0.9% NaCl) at the same volume. The renal ADC was dynamically monitored up to 40 minutes postinjection (p.i.) by DWI using a 1.5-T clinical MR unit. After DWI, the animals were killed and the kidneys were removed for iodine measurements by x-ray fluorescence analysis. RESULTS: The in vitro measurements yielded an inverse relationship between increasing viscosity and decreasing water diffusion. In vivo, a slight increase in ADC was observed immediately after administration of the low-osmolar iopromide (ΔADC=80±78 µm²/s) and saline (ΔADC=89±53 µm²/s), which normalized to the baseline level at 40 minutes p.i. In contrast, a strong decrease in ADC was observed after administration of the iso-osmolar iodixanol. This was most prominent 12 minutes p.i. (ΔADC=-555±194 µm²/s) and persisted throughout the investigation. Concomitantly, the kidney iodine concentration 50 minutes p.i. was significantly higher after iodixanol (58.6±5.3 mgI/g kidney) compared with iopromide injection (18.4±4.5 mgI/g kidney). CONCLUSION: A significant difference in the renal ADC was observed between the low-osmolar CA/saline and the iso-osmolar CA. The in vitro measurements suggest that the substantial decrease in ADC observed after administration of the iso-osmolar CA is based on the high viscosity of the agent during renal passage. This, in turn, may explain the delayed iodine retention after administration of iso-osmolar CA and demonstrates the importance of the physicochemical properties of CA during their renal elimination.

Viscosity of iodinated contrast agents during renal excretion.

OBJECTIVE: Modern iodinated non-ionic contrast agents (CAs) can be classified based on their molecular structure into monomeric and dimeric CAs and have at comparable iodine concentrations a different viscosity and osmolality. During their renal excretion, CAs are concentrated in the renal tubuli which might enhance the viscosity difference between monomeric and dimeric CAs. The viscosity of a CA might have an underestimated importance for renal safety, as suggested by recent publications. In this study, we investigated the viscosities of CAs at the concentrations expected to be present in renal tubules. This concentration process was simulated in vitro using dialysis. Furthermore, we investigated urine viscosity and urine flow in rodents after administration of several non-ionic monomeric and dimeric CAs. MATERIALS AND METHODS: To estimate the viscosity of the CAs in vivo, we performed an in vitro dialysis of monomeric and dimeric CAs at various physiological osmolalities of the renal tubulus (290, 400, 500, 700 and 1000 mOsm/kg H2O). Following the dialysis, the iodine concentrations and the viscosities of the CAs were determined. Furthermore, to investigate the concentration process in vivo, we measured the urine viscosity and the urine flow in Han Wister rats after the administration of Iopromide, Iohexol, Ioversol, Iomeprol, Iodixanol, and Iosimenol at comparable iodine concentrations. As a control, saline was injected at the same volume. RESULTS: In vitro dialysis of the dimeric CA increased the iodine concentration and strongly increased the viscosity at all tested osmolalities. In contrast, for the monomeric agents an increase in concentration and viscosity was observed only at 700 as well 1000 mOsm/kg H2O but to a lesser extent. In summary, dialysis strongly enhanced the viscosity differences between the non-ionic monomeric and dimeric CAs. The administration of dimeric CAs leads to a strong increase in urine viscosity; this was not observed for the monomeric CAs. In contrast, a significantly higher urine flow was measured after the administration of the monomeric CAs as compared to the dimeric CAs. CONCLUSION: We demonstrated that the viscosity differences between monomeric and dimeric CAs are strongly enhanced due to a concentration process of the CAs upon increasing osmolalities, a process which is likely to take place in a similar manner in the tubular system. This result suggests that the viscosity of the dimeric agents increases dramatically in vivo and gives a plausible explanation for measured enhancement of urine viscosity upon dimeric CA administration. On the other hand, the higher osmolality of the monomeric agents causes an osmodiuresis, indicated by a higher urine flow, which leads to a faster elimination of the CAs from the kidney.

The role of residual gadolinium in the induction of nephrogenic systemic fibrosis-like skin lesions in rats.

OBJECTIVE: Nephrogenic systemic fibrosis (NSF) is an acquired, idiopathic disorder. Most of the cases are observed in patients with end stage renal disease (ESRD). The objective of this nonclinical animal study was to test the hypothesis that gadolinium (Gd) deposits play a role in the induction of NSF lesions. In addition, we evaluated whether an acute response to Gd exposure can initiate a process that results in fibrosis of the skin. MATERIALS AND METHODS: Han-Wistar rats were administered 3 intravenous injections of Gd-DTPA-BMA formulated without Gd-free excess ligand (Gadodiamide without Caldiamide) at a dose of 2.5 mmol/kg of body weight (b.w.) per injection given at 24-hour or 14-, 28-, or 56-day intervals. The occurrence and development of NSF-like fibrosing dermopathy lesions were followed. The Gd concentration was determined by Inductively Coupled Plasma Mass Spectrometry in skin biopsies taken during the study and organ samples taken at the end of the study.In a separate study, after injection of a single intravenous dose of 2.5 mmol/kg b.w. Gd-DTPA-BMA administered to Han-Wistar rats, the expression of cytokines and signaling molecules in serum and skin tissue was determined by quantitative RT-PCR and Luminex technology 6 hours or 14, 28, or 56 days. RESULTS: The occurrence of NSF-like macroscopic skin lesions differed between the injection groups. Shorter injection intervals resulted in more severe skin reactions. In contrast, the injection interval did not influence the long-term presence and level of accumulation of Gd concentration in tissue. The single injection of Gd-DTPA-BMA was followed by a rapid and transient induction of signaling molecules in the serum (MCP1, MCP3, IL1, IP-10, Osteopontine SCF and Timp1) as well as in the skin (MCP1 and TGFb). CONCLUSION: The presence of NSF-like fibrosing dermopathy in rats was found to be dependent on the injection interval and not on the amount of Gd in tissue. Our findings suggest the possibility of a more acute intrinsic reaction on administration of Gd-DTPA-BMA that triggers events leading to the development of skin lesions. The finding that single injections of Gd-DTPA-BMA were accompanied by a fast and transient induction of signaling molecules that are known to be involved in several fibrotic events provides additional support for this hypothesis. The study findings, however, do not support the theory that the long-term presence of Gd plays a relevant role in the development of NSF.

Notch function in myogenesis.

Notch genes encode cell surface proteins, which are evolutionary conserved and found in invertebrates like Drosophila melanogaster as well as in all vertebrate species. The transcription factor RBP-J (Rbpsuh) is a primary nuclear mediator of Notch signals. Signals provided by Notch receptors control cell fate decisions, patterning, and they also affect proliferation or the maintenance of progenitor cells. In these Perspectives we highlight the recent findings on the role of Notch/RBP-J signaling in the maintenance of muscle progenitor cells during embryogenesis and in the generation of satellite cells in fetal development.

RBP-J (Rbpsuh) is essential to maintain muscle progenitor cells and to generate satellite cells.

In the developing muscle, a pool of myogenic progenitor cells is formed and maintained. These resident progenitors provide a source of cells for muscle growth in development and generate satellite cells in the perinatal period. By the use of conditional mutagenesis in mice, we demonstrate here that the major mediator of Notch signaling, the transcription factor RBP-J, is essential to maintain this pool of progenitor cells in an undifferentiated state. In the absence of RBP-J, these cells undergo uncontrolled myogenic differentiation, leading to a depletion of the progenitor pool. This results in a lack of muscle growth in development and severe muscle hypotrophy. In addition, satellite cells are not formed late in fetal development in conditional RBP-J mutant mice. We conclude that RBP-J is required in the developing muscle to set aside proliferating progenitors and satellite cells.

Ablation of gap junctional communication in hepatocytes of transgenic mice does not lead to disrupted cellular homeostasis or increased spontaneous tumourigenesis.

Gap junctions between murine hepatocytes are composed of two subunit proteins, connexin26 (Cx26) and connexin32 (Cx32). Previously, we found increased formation of chemically induced liver tumours but no increase in spontaneous development of preneoplastic hepatic foci in mice that lacked Cx32 and expressed decreased amounts of Cx26. In order to clarify this tumour-suppressive effect and to overcome embryonic lethality of constitutive Cx26-deficient mice, cell type-specific targeting of the Cx26 gene was performed. Mice with loxP-flanked Cx26 coding DNA were crossed with mice expressing the Cre recombinase exclusively in hepatocytes. Progeny mice lacking Cx26 in the liver were viable and fertile with no obvious signs of phenotypic alterations. To generate mice that totally lack gap junctional intercellular coupling, these mice were crossed with constitutive Cx32-deficient mice. We found no increase in spontaneously induced liver tumour formation in Cx26 and double deficient Cx26/Cx32 mice. Occasionally, double deficient livers exhibited morphological alterations, like amyloidosis, and a slightly increased basal proliferation rate of hepatocytes. Although the absence of gap junction channels led to altered expression of adhesion-related proteins like E-cadherin and actin, microarray analyses of total liver transcripts yielded only few differences between Cx26-deficient and double deficient livers compared to control samples. Our results suggest that total lack of gap junctional communication due to hepatocytic ablation of Cx26 and Cx32 does not drastically alter basal hepatocytic function and does not lead to increased spontaneous liver tumour formation.

NF-kappaB transmits Eda A1/EdaR signalling to activate Shh and cyclin D1 expression, and controls post-initiation hair placode down growth.

A novel function of NF-kappaB in the development of most ectodermal appendages, including two types of murine pelage hair follicles, was detected in a mouse model with suppressed NF-kappaB activity (c(IkappaBalphaDeltaN)). However, the developmental processes regulated by NF-kappaB in hair follicles has remained unknown. Furthermore, the similarity between the phenotypes of c(IkappaBADeltaN) mice and mice deficient in Eda A1 (tabby) or its receptor EdaR (downless) raised the issue of whether in vivo NF-kappaB regulates or is regulated by these novel TNF family members. We now demonstrate that epidermal NF-kappaB activity is first observed in placodes of primary guard hair follicles at day E14.5, and that in vivo NF-kappaB signalling is activated downstream of Eda A1 and EdaR. Importantly, ectopic signals which activate NF-kappaB can also stimulate guard hair placode formation, suggesting a crucial role for NF-kappaB in placode development. In downless and c(IkappaBalphaDeltaN) mice, placodes start to develop, but rapidly abort in the absence of EdaR/NF-kappaB signalling. We show that NF-kappaB activation is essential for induction of Shh and cyclin D1 expression and subsequent placode down growth. However, cyclin D1 induction appears to be indirectly regulated by NF-kappaB, probably via Shh and Wnt. The strongly decreased number of hair follicles observed in c(IkappaBalphaDeltaN) mice compared with tabby mice, indicates that additional signals, such as TROY, must regulate NF-kappaB activity in specific hair follicle subtypes.