Functional Magnetic Resonance Imaging of Rats with Experimental Autoimmune Encephalomyelitis Reveals Brain Cortex Remodeling.

Cortical reorganization occurring in multiple sclerosis (MS) patients is thought to play a key role in limiting the effect of structural tissue damage. Conversely, its exhaustion may contribute to the irreversible disability that accumulates with disease progression. Several aspects of MS-related cortical reorganization, including the overall functional effect and likely modulation by therapies, still remain to be elucidated. The aim of this work was to assess the extent of functional cortical reorganization and its brain structural/pathological correlates in Dark Agouti rats with experimental autoimmune encephalomyelitis (EAE), a widely accepted preclinical model of chronic MS. Morphological and functional MRI (fMRI) were performed before disease induction and during the relapsing and chronic phases of EAE. During somatosensory stimulation of the right forepaw, fMRI demonstrated that cortical reorganization occurs in both relapsing and chronic phases of EAE with increased activated volume and decreased laterality index versus baseline values. Voxel-based morphometry demonstrated gray matter (GM) atrophy in the cerebral cortex, and both GM and white matter atrophy were assessed by ex vivo pathology of the sensorimotor cortex and corpus callosum. Neuroinflammation persisted in the relapsing and chronic phases, with dendritic spine density in the layer IV sensory neurons inversely correlating with the number of cluster of differentiation 45-positive inflammatory lesions. Our work provides an innovative experimental platform that may be pivotal for the comprehension of key mechanisms responsible for the accumulation of irreversible brain damage and for the development of innovative therapies to reduce disability in EAE/MS. SIGNIFICANCE STATEMENT: Since the early 2000s, functional MRI (fMRI) has demonstrated profound modifications in the recruitment of cortical areas during motor, cognitive, and sensory tasks in multiple sclerosis (MS) patients. Experimental autoimmune encephalomyelitis (EAE) represents a reliable model of the chronic-progressive variant of MS. fMRI studies in EAE have not been performed extensively up to now. This paper reports fMRI studies in a rat model of MS with somatosensory stimulation of the forepaw. We demonstrated modifications in the recruitment of cortical areas consistent with data from MS patients. To the best of our knowledge, this is the first report of cortical remodeling in a preclinical in vivo model of MS.

Investigation of adipose tissues in Zucker rats using in vivo and ex vivo magnetic resonance spectroscopy.

In vivo single-voxel magnetic resonance spectroscopy (MRS) at 4.7T and ex vivo high-resolution proton magnetic resonance spectroscopy (HR-NMR) at 500 MHz were used to study the composition of adipose tissues in Zucker obese and Zucker lean rats. Lipid composition was characterized by unsaturation and polyunsaturation indexes and mean chain lengths. In vitro experiments were conducted in known mixtures of triglycerides and oils in order to validate the method. To avoid inaccuracies due to partial peak overlapping in MRS, peak quantification was performed after fitting of spectral peaks by using the QUEST algorithm. The intensity of different spectral lines was also corrected for T2 relaxation. Albeit with different sensitivity and accuracy, both techniques revealed that white adipose tissue is characterized by lower unsaturation and polyunsaturation indexes in obese rats compared with controls. HR-NMR revealed similar differences in brown adipose tissue. The present findings confirm the hypothesis that obese and lean Zucker rats have different adipose tissue composition.

Early versus late GD-DTPA MRI enhancement in experimental glioblastomas.

PURPOSE: To compare early versus late enhancement in two glioblastoma models characterized by different infiltrative/edematous patterns. MATERIALS AND METHODS: Three weeks after inoculation into nude mice of U87MG and U251 cells, T1-weighted images were acquired early (10.5 min), intermediate (21 min) and late (30.5 min) after a bolus injection of Gd-DTPA at 300 µ mol/kg dosage. EARLY(TH) and LATE(TH) were the corresponding volumes with an enhancement higher than a threshold TH, defined by the mean (µ) and standard deviation (σ) on a contralateral healthy area. ADD(TH) was the enhancing volume found in LATE(TH) but not in EARLY(TH). T2 imaging of both tumors was performed, and T2 mapping of U251. RESULTS: In all tumors, LATE(TH) was significantly higher than EARLY(TH) for TH ranging from µ+σ to µ+5σ. The ADD(TH) /EARLY(TH) ratio was not significantly different when U251 and U87MG tumors were compared. In the U87MG tumors, some enhancement was observed outside the regularly demarcated T2-hyperintense area. In the U251 tumors, irregularly T2 demarcated, a large portion of ADD(µ+3σ) had normal T2 values. At histology, U251 showed a higher infiltrative pattern than U87MG. CONCLUSION: In these models, the increase over time in the enhancing volume did not depend on the different infiltrative/edematous patterns and was not closely related with edema.

MRI characterization of rat brain aging at structural and functional level: Clues for translational applications.

Magnetic resonance imaging (MRI) paradigms, using non-invasive approaches, can provide relevant findings about brain aging. The attention has been primarily focused on neurodegenerative diseases, while little or nothing has been done to differentiate physiology from pathology. The present study aimed to test diffusion tensor imaging (DTI) and functional MRI (fMRI) metrics to analyze physiological age-related changes in rats at myelin structure and activation level; findings were validated by ex vivo histology. The purpose is to find comparable biomarkers in rodents and humans to allow a reliable translation from pre-clinical to clinical settings. Data evidenced: i) a significantly higher cerebrospinal fluid volume in middle-aged and aged vs. young rats; ii) a progressive alteration of white matter; iii) a significant reduction of evoked activity in aged animals. These results partially mirror the age-related changes in humans and may represent a preliminary step to find reliable tools for a lifelong monitoring with a value for the clinical practice (e.g., to provide support to the early diagnosis of dementia in asymptomatic subjects).

Washout of small molecular contrast agent in carcinoma-derived experimental tumors.

The use of contrast-enhanced magnetic resonance imaging (MRI) for the assessment of breast carcinomas reveals satisfactory sensitivity, but due to low specificity, it does not obviate the need for subsequent tissue sampling. Its capability to differentiate benign from malignant lesion is under continuous investigation. Dynamic contrast-enhanced MRI (DCE-MRI) could improve specificity of MRI through the analysis of the kinetic of contrast enhancement. In particular, the study of the washout pattern is considered a promising tool to improve in vivo diagnosis and even to evaluate the response under chemotherapy. To provide a comprehensive characterization of this parameter in malignant tumor models, in vivo mapping of the washout of small molecular contrast agent (Gd-DTPA, molecular weight 0.57 kDa) was carried out in three transplanted/spontaneous mammary tumors, which differed in their histopathological and microvascular features. It resulted that in all models around 40% of tumor volume lacks efficient washout; washout areas are frequently, but not always, restricted to the tumor periphery and that non-washout areas are not restricted to necrotic regions. Difference in the distribution of lymphatic vessels characterized spontaneous vs. transplanted tumors but did not produce a corresponding different washout pattern, confirming that Gd-DTPA drainage does not mainly depend on lymphatic architecture. Finally, the efficiency of washout is correlated with parameters obtainable during the earlier phases of the enhancement curve and in malignant tumors it could be indirectly estimated from them.

Polymer-coated superparamagnetic iron oxide nanoparticles as T2 contrast agent for MRI and their uptake in liver.

AIM: To study the efficiency of multifunctional polymer-based superparamagnetic iron oxide nanoparticles (bioferrofluids) as a T2 magnetic resonance contrast agent and their uptake and toxicity in liver. MATERIALS & METHODS: Mice were intravenously injected with bioferrofluids and Endorem®. The magnetic resonance efficiency, uptake and in vivo toxicity were investigated by means of magnetic resonance imaging (MRI) and histological techniques. RESULTS: Bioferrofluids are a good T2 contrast agent with a higher r2/r1 ratio than Endorem. Bioferrofluids have a shorter blood circulation time and persist in liver for longer time period compared with Endorem. Both bioferrofluids and Endorem do not generate any noticeable histological lesions in liver over a period of 60 days post-injection. CONCLUSION: Our bioferrofluids are powerful diagnostic tool without any observed toxicity over a period of 60 days post-injection.

Pancreatic cancer growth using magnetic resonance and bioluminescence imaging.

OBJECT: Pancreatic cancer is one of the most lethal human cancer and appropriate experimental tumor models are needed for the development of innovative therapeutic approaches. This paper describes an experimental model of human pancreatic cancer and a related non invasive imaging technique suitable for monitoring tumor growth and metastatization. The aim of the work was the implementation of an experimental platform suitable for assessing the efficacy of new therapeutic agents. MATERIALS AND METHODS: Human pancreatic cancer cells (PANC-1-Luc+) were injected into the pancreas of female athymic CD1 mice. Magnetic Resonance Imaging (MRI) at 4.7T and Bioluminescence Imaging (BLI) were performed in each mouse at three time points after cell inoculation (1, 2 and 3months). Two groups of mice were studied: the first group of n=13 mice in which 5*10(6) cells were injected and the second group of n=10 mice in which 2*10(6) cells were injected. MRI examination included T2w acquisitions and (at the last time point) Dynamic-contrast-enhanced-MRI (DCE-MRI). RESULTS: Each mouse underwent three longitudinal MRI and BLI examinations. BLI was more sensitive than MRI producing higher detection rate at early time points. Moreover in one case of abdominal dissemination of pancreatic tumor cells, small tumoral masses were detected by BLI and not detected by MRI. However BLI appears more prone to experimental error most likely due to photon attenuation. In 4 mice BLI produced false negative results. DCE-MRI experiments providing information on tumor perfusion were conducted successfully in this anatomical district and demonstrated that the tumor tissues from the second experimental group are more vascularized compared to the first group. CONCLUSION: The present study performed on the experimental model of pancreatic cancer here described shows that MRI and BLI are complementary techniques and that synergistic application of both can overcome the intrinsic limitations of each.

Manganese-enhanced magnetic resonance imaging investigation of the interferon-α model of depression in rats.

Therapeutic effects of interferon-α (IFN-α) are known to be associated with CNS toxicity in humans, and in particular with depression symptoms. Animal models of IFN-α-induced depression (sickness behaviour) have been developed in rodents using various preparations, dosing schedules or routes of administrations. In this work, Manganese Enhanced MRI (MEMRI) has been applied to investigate an experimental model of sickness behaviour induced by administration of IFN-α in rats. IFN-α (3.10(5) U/kg), or vehicle, was daily administered i.p., for 7days in rats (n=20 IFN-α treated and n=20 controls). After treatment, animals were assigned to behavioural (n=10 treated, n=10 control) or MRI (n=10 treated and n=10 control) studies. Animals assigned to the MRI study received two repeated i.p. injections of MnCl2, before image acquisition. Images were acquired at 4.7T using T1 mapping for determination of Mn concentration in brain. After co-registration of T1 maps to a digital brain atlas, differences between brains of treated and untreated animals were assessed pixel-to-pixel by statistical analysis. Behavioural tests showed alterations in freezing and struggling parameters, as expected in an experimental model of sickness behaviour. MRI showed a well defined brain region, mainly contained in the visual cortex, in which Mn uptake was significantly lower in treated than in control animals, indicating probably altered functionality. No significant difference was detected in other brain regions. In addition, a statistically significant decrease in the volume of the pituitary gland, paralleled by a slight increase in its Mn content, was detected in treated animals. MEMRI provides both morphological and functional information in the brain of small laboratory animals and can constitute a valuable tool in the investigation of experimental models of psychiatric diseases.

In vivo long-term magnetic resonance imaging activity of ferritin-based magnetic nanoparticles versus a standard contrast agent.

New long-circulating maghemite nanoparticles of 4 and 6 nm, coated with an apoferritin protein capsid, exhibit useful properties to act as magnetic resonance imaging (MRI) contrast agents. A full in vivo study of the so-called apomaghemites reveals that their long-term MRI properties are better than those of a standard superparamagnetic iron oxide (SPIO) widely used in biomedical applications. The biodistribution of apomaghemites and standard SPIO was investigated by MRI in mice at two different concentrations, 6 and 2.5 mg of Fe·kg(-1), over 60 days. Significant differences are found at low dose (2.5 mg of Fe·kg(-1)). Thus, whereas apomaghemites are active for MR bioimaging of liver for 45 days, standard SPIO is not effective beyond 7 days. On the basis of our data, we may concluded that apomaghemites can act as new long-term MRI liver contrast agents, allowing first the diagnosis of a liver pathology and then monitoring after treatment without the need for a second injection.

Modulation of fronto-cortical activity by modafinil: a functional imaging and fos study in the rat.

Modafinil (MOD) is a wake-promoting drug with pro-cognitive properties. Despite its increasing use, the neuronal substrates of MOD action remain elusive. In particular, animal studies have highlighted a putative role of diencephalic areas as primary neuronal substrate of MOD action, with inconsistent evidence of recruitment of fronto-cortical areas despite the established pro-cognitive effects of the drug. Moreover, most animal studies have employed doses of MOD of limited clinical relevance. We used pharmacological magnetic resonance imaging (phMRI) in the anesthetized rat to map the circuitry activated by a MOD dose producing clinically relevant plasma exposure, as here ascertained by pharmacokinetic measurements. We observed prominent and sustained activation of the prefrontal and cingulate cortex, together with weaker but significant activation of the somatosensory cortex, medial thalamic domains, hippocampus, ventral striatum and dorsal raphe. Correlation analysis of phMRI data highlighted enhanced connectivity within a neural network including dopamine projections from the ventral tegmental area to the nucleus accumbens. The pro-arousing effect of MOD was assessed using electroencephalographic recording under anesthetic conditions comparable to those used for phMRI, together with the corresponding Fos immunoreactivity distribution. MOD produced electroencephalogram desynchronization, resulting in reduced delta and increased theta frequency bands, and a pattern of Fos induction largely consistent with the phMRI study. Altogether, these findings show that clinically relevant MOD doses can robustly activate fronto-cortical areas involved in higher cognitive functions and a network of pro-arousing areas, which provide a plausible substrate for the wake-promoting and pro-cognitive effects of the drug.

DCE-MRI using small-molecular and albumin-binding contrast agents in experimental carcinomas with different stromal content.

OBJECTIVES: To compare DCE-MRI experiments performed using a standard small-molecular (Gd-DTPA) and an albumin-binding (MS-325) contrast agent in two carcinoma models with different stromal content. MATERIALS AND METHODS: DU-145 or BXPC-3 cancer cells were subcutaneously injected into nude mice. DCE-MRI was performed by a bolus injection of Gd-DTPA or MS-325 about 2 weeks after inoculation. For quantitative analysis a volume of interest was manually drawn over each tumor. To address the heterogeneous enhancement, each tumor volume was then divided into the 20% most-enhancing and the remaining 80% least-enhancing fractions. Mean tumor enhancement was calculated over these selected tumor volumes and compared between tumor groups and contrast agents. Maps of differential enhancement, peak enhancement and time-to-peak were used for visual evaluation. CD31 and VEGF immunohistochemistry were performed in excised tumors. RESULTS: In the 80% least-enhancing volume, at late time points of the dynamic scan, the mean enhancement elicited by MS-325 was higher in BXPC-3 than in DU-145 tumors. In the 20% most-enhancing volume, using either contrast agents, significant difference between the two tumors types were observed only early, while at later time points of the dynamic scan the difference were obscured by the faster washout observed in the BXPC-3 tumors. Enhancement maps confirmed that BXPC-3 tumors were characterized by marked washout rate using either contrast agent, particularly in the higher enhancing peripheral rim. With MS-325 this washout pattern appeared to be specific to the BXPC-3 carcinomas, since it was not observed in the DU-145 tumors. Finally, in both tumor types, MS-325 produced significantly higher enhancement than Gd-DTPA in the late phase of the dynamic scan. Ex vivo analysis confirmed the marked presence of aberrant infiltrative stroma in BXPC-3 tumors, in which tumor vessels were embedded. In all tumors the central portion was less viable and less infiltrated by stromal tissue then the peripheral areas. CONCLUSIONS: Contrast distribution proved to be related to stromal content, which presumably produced the higher enhancement and faster washout observed in the BXPC-3 tumors. In particular, 'early' contrast-enhanced MRI, appeared as the most sensitive technique to detect the tumor portions characterized by a high stromal content, i.e. the peripheral rim of the BXPC-3 tumors. Since the same tumor models were recently investigated using FDG-PET imaging, showing inverse relationship between FDG uptake and stromal content, contrast-enhanced MRI and FDG-PET could provide complementary and comprehensive sensitivity in the assessment of carcinomas.

Evaluation of cryopreserved donor skin viability: the experience of the regional tissue bank of Verona.

BACKGROUND: Allogeneic human skin removed from cadaveric donors is the covering of choice for deep burns, since it accelerates the re-epithelialisation of autologous skin. In this study we evaluated the cellular viability of cryopreserved skin at the regional tissue bank of Verona (Italy). METHODS: From 1st June 2007 to 30th September 2007, tests of cutaneous cell viability were carried out on 21 consecutive skin donors using the MTT (tetrazolium salt) method on samples prior to freezing and on thawed samples after a period of cryopreservation. RESULTS: The mean percentage viability was 45.1% (+/-20.1%), which is similar to results obtained in other tissue banks. It was noted that viability decreased with increasing age of the donor. CONCLUSIONS: The results of the evaluation of cutaneous cell viability document the validity of the skin cryopreservation procedure in use at the tissue bank in Verona.

In vivo visualization of transplanted pancreatic islets by MRI: comparison between in vivo, histological and electron microscopy findings.

The aim of the work was to compare in vivo MRI visualization of pancreatic islets labeled with clinical-grade superparamagnetic iron oxide (SPIOs) contrast agents with ex vivo examination of liver tissue in an experimental model of marginal mass transplantation in rats. Seven hundred IEq (Islet Equivalent) from Wistar rats, labeled by incubation with Endorem or Resovist, were transplanted into Sprague-Dawley rats through the portal vein. Liver MR images of recipient rats were acquired at different time points (3-42 days) after transplantation. Animals were sacrificed during this period and their livers were excised and prepared for histology and electron microscopy. Hypointense spots originating from iron particles were observed in MR images. The number of separate spots was counted. Three days after transplantation one spot for every three or four transplanted islets was observed. Seven days after transplantation, histological sections showed the presence of iron within pancreatic islets. The time course of MR images showed a decrease in the number of spots, at 42 days, amounting to 65 and 22% of the initial value, for Resovist and Endorem respectively, while no immunopositive endocrine cells were detected in histological slices. The present work shows that pancreatic islets can be labeled using clinically approved SPIO contrast agents and visualized using in vivo MRI with high sensitivity, consistently with findings in the literature. Differently from reports in the literature, our findings indicate that iron particles could last in the liver for long periods, independently of the presence of intact pancreatic islets.

Tumor microvasculature observed using different contrast agents: a comparison between Gd-DTPA-Albumin and B-22956/1 in an experimental model of mammary carcinoma.

OBJECTIVE: The aim of this study was to compare a pure macromolecular contrast agent (Gd-DTPA-albumin) with a new protein-binding blood pool contrast agent (B22956/1) in terms of their capacity to investigate the microvasculature in an experimental model of mammary carcinoma. MATERIALS AND METHODS: Tumors were induced by subcutaneous injection of 5 x 10(5) BB1 cells into the backs of 5-7 week-old female FVB/neuNT233 mice. The animals were observed using DCE-MRI when the longest diameter of the tumor was 10.2+/-2.0 mm. DCE-MRI experiments were carried out using B22956/1 and (24 h later) Gd-DTPA-albumin. RESULTS: DCE-MRI data showed that vasculature in the tumor rim was characterized by greater fractional plasma volume and transendothelial permeability than vasculature in the tumor core as measured by both contrast agents. Permeability to Gd-DTPA-albumin in the tumor core was hardly measurable while permeability to B22956/1 was substantial. Histologically the tumor core showed areas of well vascularized, viable tissue surrounded by necrotic regions. CONCLUSIONS: DCE-MRI experiments performed with B22956/1 are useful in the investigation of vasculature in those tumor regions that are characterized by low permeability to macromolecules.