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Starting from the mid-1990s, several iron oxide nanoparticles (NPs) were developed as MRI contrast agents. Since their sizes fall in the tenths of a nanometer range, after i.v. injection these NPs are preferentially captured by the reticuloendothelial system of the liver. They have therefore been proposed as liver-specific contrast agents. Even though their unfavorable cost/benefit ratio has led to their withdrawal from the market, innovative applications have recently prompted a renewal of interest in these NPs. One important and innovative application is as diagnostic agents in cancer immunotherapy, thanks to their ability to track tumor-associated macrophages (TAMs) in vivo. It is worth noting that iron oxide NPs may also have a therapeutic role, given their ability to alter macrophage polarization. This review is devoted to the most recent advances in applications of iron oxide NPs in tumor diagnosis and therapy. The intrinsic therapeutic effect of these NPs on tumor growth, their capability to alter macrophage polarization and their diagnostic potential are examined. Innovative strategies for NP-based drug delivery in tumors (e.g., magnetic resonance targeting) will also be described. Finally, the review looks at their role as tracers for innovative, and very promising, imaging techniques (magnetic particle imaging-MPI).

Porous silicon (pSi) microparticles obtained by porosification of crystalline silicon wafers have unique optical properties that, together with biodegradability, biocompatibility and absence of immunogenicity, are fundamental characteristics to candidate them as tracers in optical imaging techniques and as drug carriers. In this work, we focus on the possibility to track down the pSi microparticles also by MRI (magnetic resonance imaging), thus realizing a comprehensive tool for theranostic applications, i.e., the combination of therapy and diagnostics. We have developed and tested an easy, quick and low-cost protocol to infiltrate the COOH-functionalized pSi microparticles pores (tens of nanometers about) with magnetic nanospheres (SPIONs—Super Paramagnetic Iron Oxide Nanoparticles, about 5−7 nm) and allow an electrostatic interaction. The structural properties and the elemental composition were investigated by electron microscopy techniques coupled to elemental analysis to demonstrate the effective attachment of the SPIONs along the pores’ surface of the pSi microparticles. The magnetic properties were investigated under an external magnetic field to determine the relaxivity properties of the material and resulting in an alteration of the relaxivity of water due to the SPIONs presence, clearly demonstrating the effectiveness of the easy functionalization protocol proposed.

Localization, differentiation and quantitative assessment of fat tissues have always collected the interest of researchers. Nowadays, these topics are even more relevant as obesity (the excess of fat tissue) is considered a real pathology requiring in some cases pharmacological and surgical approaches. Several weight loss medications, acting either on the metabolism or on the central nervous system, are currently under preclinical or clinical investigation. Animal models of obesity have been developed which are widely used in pharmaceutical research. The assessment of candidate drugs in animal models requires non-invasive methods for longitudinal assessment of efficacy, the main outcome being the amount of body fat. Fat tissues can be either quantified in the entire animal or localized and measured in selected organs/regions of the body. Fat tissues are characterized by peculiar contrast in several imaging modalities as for example Magnetic Resonance Imaging (MRI) that can distinguish between fat and water protons thank to their different magnetic resonance properties. Since fat tissues have higher carbon/hydrogen content than other soft tissues and bones, they can be easily assessed by Computed Tomography (CT) as well. Interestingly, MRI also discriminates between white and brown adipose tissue; the latter has long been regarded as a potential target for anti-obesity drugs because of its ability to enhance energy consumption through increased thermogenesis. Positron Emission Tomography (PET) performed with 18F-FDG as glucose analogue radiotracer reflects well the metabolic rate in body tissues and consequently is the technique of choice for studies of BAT metabolism. This review will focus on the main, non-invasive imaging techniques (MRI, CT and PET) that are fundamental for the assessment, quantification and functional characterization of fat deposits in small laboratory animals. The contribution of optical techniques, which are currently regarded with increasing interest, will be also briefly described. For each technique the physical principles of signal detection will be overviewed and some relevant studies will be summarized. Far from being exhaustive, this review has the purpose to highlight some strategies useful for the in vivo identification, quantification and functional characterization of adipose tissues mainly from the point of view of biophysics and physiology.

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.

Glucose transport and consumption are increased in tumors, and this is considered a diagnostic index of malignancy. However, there is recent evidence that carcinoma-associated stromal cells are capable of aerobic metabolism with low glucose consumption, at least partly because of their efficient vascular supply. In the present study, using dynamic contrast-enhanced magnetic resonance imaging and [F-18]fluorodeoxyglucose (FDG) positron emission tomography (PET), we mapped in vivo the vascular supply and glucose metabolism in syngeneic experimental models of carcinoma and mesenchymal tumor. We found that in both tumor histotypes, regions with high vascular perfusion exhibited a significantly lower FDG uptake. This reciprocity was more conspicuous in carcinomas than in mesenchymal tumors, and regions with a high-vascular/low-FDG uptake pattern roughly overlapped with a stromal capsule and intratumoral large connectival septa. Accordingly, mesenchymal tumors exhibited a higher vascular perfusion and a lower FDG uptake than carcinomas. Thus, we provide in vivo evidence of vascular/metabolic reciprocity between epithelial and mesenchymal histotypes in tumors, suggesting a new intriguing aspect of epithelial-stromal interaction. Our results suggests that FDG-PET-based clinical analysis can underestimate the malignity or tumor extension of carcinomas exhibiting any trait of “mesenchymalization” such as desmoplasia or epithelial-mesenchymal transition.

Contrast-enhanced ultrasound (CEUS) is an advanced approach to in vivo assessment of tumor vascularity and is being increasingly adopted in clinical oncology. It is based on 1- to 10 μm-sized gas microbubbles, which can cross the capillary beds of the lungs and are effective echo enhancers. It is known that high cell density, high transendothelial fluid exchange, and poorly functioning lymphatic circulation all provoke solid stress, which compresses vessels and drastically reduces tumor blood flow. Given their size, we supposed that the perfusion of microbubbles is affected by anatomic features of tumor vessels more than are contrast agents traditionally used in dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). Here, we compared dynamic information obtained from CEUS and DCE-MRI on two experimental tumor models exhibiting notable differences in vessel anatomy. We found that tumors with small, flattened vessels show a much higher resistance to microbubble perfusion than to MRI contrast agents, and appear scarcely vascularized at CEUS examination, despite vessel volume adequate for normal function. Thus, whereas CEUS alone could induce incorrect diagnosis when tumors have small or collapsed vessels, integrated analysis using CEUS and DCE-MRI allows in vivo identification of tumors with a vascular profile frequently associated with malignant phenotypes.

Magnetic nanoparticles (MNPs) are capable of generate heating power under the influence of alternating magnetic fields (AMF); this behaviour recently opened new scenarios for advanced biomedical applications, mainly as new promising tumor therapies. In this paper we have tested magnetic nanoparticles called magnetosomes (MNs): a class of MNPs naturally produced by magnetotactic bacteria. We extracted MNs from Magnetospirillum gryphiswaldense strain MSR-1 and tested the interaction with cellular elements and anti-neoplastic activity both in vitro and in vivo, with the aim of developing new therapeutic approaches for neoplastic diseases. In vitro experiments performed on Human Colon Carcinoma HT-29 cell cultures demonstrated a strong uptake of MNs with no evident signs of cytotoxicity and revealed three phases in the interaction: adherence, transport and accumulation in Golgi vesicles. In vivo studies were performed on subcutaneous tumors in mice; in this model MNs are administered by direct injection in the tumor volume, then a protocol consisting of three exposures to an AMF rated at 187 kHz and 23kA/m is carried out on alternate days, over a week. Tumors were monitored by Magnetic Resonance Imaging (MRI) to obtain information about MNs distribution and possible tissue modifications induced by hyperthermia. Histological analysis showed fibrous and necrotic areas close to MNs injection sites in mice subjected to a complete thermotherapy protocol. These results, although concerning a specific tumor model, could be useful to further investigate the feasibility and efficacy of protocols based on MFH. Magnetic nanoparticles naturally produced and extracted from bacteria seem to be promising candidates for theranostic applications in cancer therapy.

Graft vascularization is a crucial step to obtain stable normoglycemia in pancreatic islet transplantation. Endothelial progenitor cells (EPCs) contribute to neoangiogenesis and to the revascularization process during ischaemic events and play a key role in the response to pancreatic islet injury. In this work we co-transplanted EPCs and islets in the portal vein of chemically-induced diabetic rats to restore islet vascularization and to improve graft survival. Syngenic islets were transplanted, either alone or with EPCs derived from green fluorescent protein (GFP) transgenic rats, into the portal vein of streptozotocin-induced diabetic rats. Blood glucose levels were monitored and intraperitoneal glucose tolerance tests were performed. Real time-PCR was carried out to evaluate the gene expression of angiogenic factors. Diabetic-induced rats showed long-lasting (6 months) normoglycemia upon co-transplantation of syngenic islets and EPCs. After 3-5 days from transplantation, hyperglycaemic levels dropped to normal values and lasted unmodified as long as they were checked. Further, glucose tolerance tests revealed the animals' ability to produce insulin on-demand as indexed by a prompt response in blood glucose clearance. Graft neovascularization was evaluated by immunohistochemistry: for the first time the measure of endothelial thickness revealed a donor-EPC-related neovascularization supporting viable islets up to six months after transplant. Our results highlight the importance of a newly formed viable vascular network together with pancreatic islets to provide de novo adequate supply in order to obtain enduring normoglycemia and prevent diabetes-related long-term health hazards.

In the present paper, chemical shift imaging techniques are applied to quantitative in vivo evaluation of fat and water content in interscapular brown adipose tissue (BAT). The experiments have been carried out on five female Sprague-Dawley rats after calibration and testing with suitable phantoms containing known amounts of water and oil. We found that, in the interscapular BAT, the fat is about 50% at the surface (mainly unilocular) region, but its percentage drops to 20–30% in the deepest (mainly multilocular) portion. The perirenal deposits of white adipose tissue (WAT) contained significantly higher amount of fat with large areas ranging from 70 to 90%. Later the rats were killed and the same procedure was repeated with dead animals. Experiments performed in dead rats show a modification of the hydro-lipidic ratio more evident in the multilocular portions of the deposit. The present work demonstrates that MRI-based methods allow a non-invasive, in vivo quantitative mapping of the lipid content which can be applied to investigation of brown adipose tissue deposits in small experiment animals.—Lunati, E., P. Marzola, E. Nicolato, M. Fedrigo, M. Villa, and A. Sbarbati. In vivo quantitative lipidic map of brown adipose tissue by chemical shift imaging at 4.7 tesla. J. Lipid Res. 1999. 40: 1395–1400.

Pilocarpine-induced seizures in rats provide a widely animal model of temporal lobe epilepsy. Some evidences reported in the literature suggest that at least 1 h of status epilepticus (SE) is required to produce subsequent chronic phase, due to the SE-related acute neuronal damage. However, recent data seems to indicate that neuro-inflammation plays a crucial role in epileptogenesis, modulating secondarily a neuronal insult. For this reason, we decided to test the following hypotheses: a) whether pilocarpine-injected rats that did not develop SE can exhibit long-term chronic spontaneous recurrent seizures (SRS) and b) whether acute neurodegeneration is mandatory to obtain chronic epilepsy. Therefore, we compared animals injected with the same dose of pilocarpine that developed or did not SE, and saline treated rats. We used telemetric acquisition of EEG as long-term monitoring system to evaluate the occurrence of seizures in non-SE pilocarpineinjected animals. Furthermore, histology and MRI analysis were applied in order to detect neuronal injury and neuropathological signs. Our observations indicate that non-SE rats exhibit SRS almost 8 (+/22) months after pilocarpine-injection, independently to the absence of initial acute neuronal injury. This is the first time reported that pilocarpine injected rats without developing SE, can experience SRS after a long latency period resembling human pathology. Thus, we strongly emphasize the important meaning of including these animals to model human epileptogenesis in pilocarpine induced epilepsy.

Growing evidences indicate that proteases are implicated in adipogenesis and in the onset of obesity. We previously reported that the cysteine protease cathepsin K (ctsk) is overexpressed in the white adipose tissue (WAT) of obese individuals. We herein characterized the WAT and the metabolic phenotype of ctsk deficient animals (ctsk-/-). When the growth rate of ctsk-/- was compared to that of the wild type animals (WT), we could establish a time window (5-8 weeks of age) within which ctsk-/-display significantly lower body weight and WAT size as compared to WT. Such a difference was not observable in older mice. Upon treatment with high fat diet (HFD) for 12 weeks ctsk-/- gained significantly less weight than WT and showed reduced brown adipose tissue, liver mass and a lower percentage of body fat. Plasma triglycerides, cholesterol and leptin were significantly lower in HFD-fed-ctsk-/- as compared to HFD-fed WT animals. Adipocyte lipolysis rates were increased in both young and HFD-fed-ctsk-/-, as compared to WT. Carnitine palmitoyl transferase-1 activity, was higher in mitochondria isolated from the WAT of HFD treated ctsk-/- as compared to WT. Together, these data indicate that ctsk ablation in mice results in reduced body fat content under conditions requiring a rapid accumulation of fat stores. This observation could be partly explained by an increased release and/or utilization of FFA and by an augmented ratio of lipolysis/lipogenesis. These results also demonstrate that under a HFD, ctsk deficiency confers a partial resistance to the development of dyslipidemia.

The neuron loss characteristic of hippocampal sclerosis in temporal lobe epilepsy patients is thought to be the result of excitotoxic, rather than ischemic, injury. In this study, we assessed changes in vascular structure, gene expression, and the time course of neuronal degeneration in the cerebral cortex during the acute period after onset of pilocarpine-induced status epilepticus (SE). Immediately after 2 hr SE, the subgranular layers of somatosensory cortex exhibited a reduced vascular perfusion indicative of ischemia, whereas the immediately adjacent supragranular layers exhibited increased perfusion. Subgranular layers exhibited necrotic pathology, whereas the supergranular layers were characterized by a delayed (24 h after SE) degeneration apparently via programmed cell death. These results indicate that both excitotoxic and ischemic injuries occur during pilocarpine-induced SE. Both of these degenerative pathways, as well as the widespread and severe brain damage observed, should be considered when animal model-based data are compared to human pathology.