Lorenzo Di Cesare Mannelli

A Smart Platform for Hyperthermia Application in Cancer Treatment: Cobalt-Doped Ferrite Nanoparticles Mineralized in Human Ferritin Cages

Magnetic nanoparticles, MNPs, mineralized within a human ferritin protein cage, HFt, can represent an appealing platform to realize smart therapeutic agents for cancer treatment by drug delivery and magnetic fluid hyperthermia, MFH. However, the constraint imposed by the inner diameter of the protein shell (ca. 8 nm) prevents its use as heat mediator in MFH when the MNPs comprise pure iron oxide. In this contribution, we demonstrate how this limitation can be overcome through the controlled doping of the core with small amount of Co(II). Highly monodisperse doped iron oxide NPs with average size of 7 nm are mineralized inside a genetically modified variant of HFt, carrying several copies of α-melanocyte-stimulating hormone peptide, which has already been demonstrated to have excellent targeting properties toward melanoma cells. HFt is also conjugated to poly(ethylene glycol) molecules to increase its in vivo stability. The investigation of hyperthermic properties of HFt-NPs shows that a Co doping of 5% is enough to strongly enhance the magnetic anisotropy and thus the hyperthermic efficiency with respect to the undoped sample. In vitro tests performed on B16 melanoma cell line demonstrate a strong reduction of the cell viability after treatment with Co doped HFt-NPs and exposure to the alternating magnetic field. Clear indications of an advanced stage of apoptotic process is also observed from immunocytochemistry analysis. The obtained data suggest this system represents a promising candidate for the development of a protein-based theranostic nanoplatform.

Oxaliplatin-Induced Neuropathy: Oxidative Stress as Pathological Mechanism. Protective Effect of Silibinin

UNLABELLED Oxaliplatin is the standard treatment for advanced colorectal cancer. Its dose-limiting toxicity is the development of a painful neuropathic syndrome sustained by unclear mechanisms. Although the oxidative hypothesis is a matter of debate, direct data about oxidative damage induced in vivo by anticancer agents are lacking and the efficacy of the available antioxidant compounds are unsatisfactory. In a rat model of painful oxaliplatin-induced neuropathy (2.4 mgkg(-1) i.p., daily for 21 days), we described an important component of oxidative stress. In the plasma of oxaliplatin-treated rats, the increases in carbonylated protein and thiobarbituric acid reactive substances were the index of the resultant protein oxidation and lipoperoxidation, respectively. The same pattern of oxidation was revealed also in the sciatic nerve, and in the spinal cord where the damage reached the DNA level. The antioxidant compound silibinin (100 mgkg(-1) per os), administered once a day, starting from the first day of oxaliplatin injection until the 20th, prevented oxidative damage as did α-tocopherol. Repetitive administration of silibinin, as well as α-tocopherol, reduced oxaliplatin-dependent pain induced by mechanical and thermal stimuli. Antioxidants were also able to improve motor coordination. The antineuropathic effect of both molecules improved by about 50% oxaliplatin-induced behavioral alterations. PERSPECTIVE This study characterizes oxidative stress parameters in a rat model of oxaliplatin-induced neuropathy. A relationship between the improvement of oxidative alterations and pain relief is established in rats treated with natural antioxidant compounds like α-tocopherol and silibinin. Silibinin could be a valid therapeutic option for chemotherapy-induced neuropathy.

Morphologic Features and Glial Activation in Rat Oxaliplatin-Dependent Neuropathic Pain

UNLABELLED Neurotoxicity is the limiting side effect of the anticancer agent oxaliplatin. A tangled panel of symptoms, sensory loss, paresthesia, dysesthesia, and pain may be disabling for patients and adversely affect their quality of life. To elucidate the morphologic and molecular alterations that occur in the nervous system during neuropathy, rats were daily injected with 2.4 mg kg(-1) oxaliplatin intraperitoneally. A progressive decrease in the pain threshold and hypersensitivity to noxious and nonnoxious stimuli were evidenced during the treatment (7, 14, 21 days). On day 21, morphometric alterations were detectable exclusively in the dorsal root ganglia, whereas the activating transcription factor 3 and neurofilament (heavy-chain) expression changed dramatically in both the nerves and ganglia. Inflammatory features were not highlighted. Interestingly, satellite cells exhibited signs of activation. Glial modulation was characterized in the spinal cord and brain areas involved in pain signaling. On the 21st day, spinal astrocytes increased numerically whereas the microglial population was unaltered. The number of glial cells in the brain differed according to the zone and treatment time points. In particular, on day 21, a significant astrocyte increase was measured in the anterior cingulate cortex, somatosensory area 1, neostriatum, ventrolateral periaqueductal gray, and nucleus raphe magnus. PERSPECTIVES These data highlight the relevance of glial cells in chemotherapy-induced neurotoxicity as part of the investigation of the role that specific brain areas play in neuropathy.

Glial role in oxaliplatin-induced neuropathic pain

Oxaliplatin, a platinum-based chemotherapeutic agent, has become a standard treatment for advanced colorectal cancer. The dose-limiting toxicity of this compound is the development of peripheral neuropathy. A tangled panel of symptoms, sensory loss, paresthesia, dysesthesia and pain, may be disabling for patients and adversely affect their quality of life. Recently, we described a characteristic glial activation profile in a rat model of oxaliplatin-induced neuropathy. Glial cells are considered a new pharmacological target for neuropathic pain relief but its relevance in chemotherapy-dependent neuropathies is debated. Aimed to evaluate the significance of glial activation in pain generated by oxaliplatin, the microglial inhibitor minocycline or the astrocyte inhibitor fluorocitrate were continuously infused by intrathecal route in oxaliplatin-treated rats. Both compounds significantly reduced oxaliplatin-evoked pain though the efficacy of fluorocitrate was higher revealing a prominent role of astrocytes. Immunohistochemical analysis of the dorsal horn confirmed the specific Iba1-positive cell inhibition caused by minocycline as well as the selectivity of fluorocitrate on GFAP-positive cells. The activation of astrocytes in minocycline-treated rats suggests a microglia-independent modulation of astrocytes by oxaliplatin neurotoxicity. Neither the selective activation of astrocyte after minocycline treatment nor the exclusive microglial response after fluorocitrate is able to evoke pain. Morphometric and morphological determinations performed on dorsal root ganglia evidenced that the glial inhibitors did not prevent the oxaliplatin-dependent increase of eccentric nucleoli and multinucleolated neurons. The decrease of soma area was also unaltered. In summary, these data highlight the role of central glial cells in oxaliplatin-dependent neuropathic pain. On the other hand, glial inhibition is not associated with neuroprotective effects suggesting the need for careful modulation of glial signaling to prevent the pathophysiology that leads to persistent neuropathic pain.

A class of sulfonamide carbonic anhydrase inhibitors with neuropathic pain modulating effects

A series of benzene sulfonamide carbonic anhydrase (CA, EC 4.2.1.1) inhibitors which incorporate lipophilic 4-alkoxy- and 4-aryloxy moieties, together with several derivatives of ethoxzolamide and sulfanilamide are reported. These derivatives were investigated as inhibitors of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) of which multiple isoforms are known, and some appear to be involved in pain. These sulfonamides showed modest inhibition against the cytosolic isoform CA I, but were generally effective, low nanomolar CA II, VII, IX and XII inhibitors. X-ray crystallographic data for the adduct of several such sulfonamides with CA II allowed us to rationalize the good inhibition data. In a mice model of neuropathic pain induced by oxaliplatin, one of the strong CA II/VII inhibitors reported here induced a long lasting pain relieving effect, a fact never observed earlier. This is the first report of rationally designed sulfonamide CA inhibitors with pain effective modulating effects.

Protective effect of acetyl-l-carnitine on the apoptotic pathway of peripheral neuropathy

Peripheral neuropathies are widespread disorders induced by autoimmune diseases, drug or toxin exposure, infections, metabolic insults or trauma. Nerve damage may cause muscle weakness, altered functionalities and sensitivity, and a chronic pain syndrome characterized by allodynia and hyperalgesia. Pathophysiological mechanisms related to neuropathic disease are associated with mitochondrial dysfunctions that lead to the activation of the apoptotic cascade. In a model of peripheral neuropathy, obtained by the loose ligation of the rat sciatic nerve (CCI), we describe a nerve apoptotic state that encompasses the release of cytochrome C in the cytosol, the activation of caspase 3, and the fragmentation of the genome. Animal treatment with acetyl‐l‐carnitine (ALCAR), but not with l‐carnitine (L‐Carn) or Gabapentin, prevents apoptosis induction. ALCAR reduces cytosolic cytochrome C and caspase 3 active fragments expression in a significant manner with respect to saline treatment. Accordingly, ALCAR treatment impairs caspase 3 protease activity, as demonstrated by reduced levels of cleaved PARP. Finally, ALCAR decreases the number of piknotic nuclei. This protection correlates with the induction of X‐linked inhibitor apoptosis protein (XIAP). Taken together these results show that CCI is a valuable model to investigate neuropathies‐related apoptosis phenomena and that ALCAR is able to prevent regulated cell death in the damaged sciatic nerve.

α-Conotoxin RgIA protects against the development of nerve injury-induced chronic pain and prevents both neuronal and glial derangement

&NA; &agr;‐Conotoxin RgIA prevents the development of chronic pain, halts progression of damage in injured nerves, and limits downstream pathological alterations in dorsal root ganglion and spinal cord. &NA; Neuropathic pain affects millions of people worldwide, causing substantial disability and greatly impairing quality of life. Commonly used analgesics or antihyperalgesic compounds are generally characterized by limited therapeutic outcomes. Thus, there is a compelling need for novel therapeutic strategies able to prevent nervous tissue alterations responsible for chronic pain. The &agr;9&agr;10 nicotinic acetylcholine receptor antagonist &agr;‐conotoxin RgIA (RgIA), a peptide isolated from the venom of a carnivorous cone snail, induces relief in both acute and chronic pain models. To evaluate potential disease‐modifying effects of RgIA, the compound was given to rats following chronic constriction injury (CCI) of the sciatic nerve. Two or 10 nmol RgIA injected intramuscularly once a day for 14 days reduced the painful response to suprathreshold stimulation, increased pain threshold to nonnoxious stimuli, and normalized alterations in hind limb weight bearing. Histological analysis of the sciatic nerve revealed that RgIA prevented CCI‐induced decreases of axonal compactness and diameter, loss of myelin sheath, and decreases in the fiber number. Moreover, RgIA significantly reduced edema and inflammatory infiltrate, including a decrease of CD86+ macrophages. In L4‐L5 dorsal root ganglia, RgIA prevented morphometric changes and reduced the inflammatory infiltrate consistent with a disease‐modifying effect. In the dorsal horn of the spinal cord, RgIA prevented CCI‐induced activation of microglia and astrocytes. These data suggest that RgIA‐like compounds may represent a novel class of therapeutics for neuropathic pain that protects peripheral nervous tissues as well as prevents central maladaptive plasticity by inhibiting glial cell activation.

The Influence of Diffusion-and Relaxation-Related Factors on Signal Intensity : An Introductive Guide to Magnetic Resonance Diffusion-Weighted Imaging Studies

In magnetic resonance diffusion-weighted imaging, signal intensity is influenced simultaneously by temperature, diffusivity, b value, pseudodiffusion, macroscopic motion, and T2-weighted intensity value. The purpose of this pictorial essay is to discuss and exemplify the influence that such factors and the related modifications have on signal intensity. Apparent diffusion coefficient, shine-through and pseudodiffusion will also be examined to show how T2-weighted signal intensity value and nondiffusional intravoxel incoherent motion can affect the diffusion-weighted imaging.

Protective effect of alpha7 nAChR: Behavioural and morphological features on neuropathy

&NA; Traumatic, toxic or metabolic damage to the nervous system is the etiological foundation of neuropathic pain. Neuropathies are extremely difficult to treat and available drugs rarely joint an anti‐hyperalgesic with a neurorestorative effect. From the literature, evidences support the alpha7 nicotinic receptor (nAChR) subtype as having a role in neuropathic pain as well as possessing neuroprotective properties. Aimed to inquire the anti‐neuropathic effect of the alpha7 nAChR stimulation, we evaluated the pharmacological profile of the alpha7 nAChR agonist PNU‐282987 on pain and on morphological alterations induced in the rat sciatic nerve by loose ligation (CCI). Acute administration of PNU‐282987, 10 and 30 mg kg−1 p.o. (15 days after ligation), was able to reduce hyperalgesia in a methyllicaconitine‐reversed manner. This alpha7 nAChR agonist exerted no analgesic effects. Chronic PNU‐282987 treatments, 30 mg kg−1 once a day for 7 days and 10 mg kg−1 for 28 days, were able to decrease pain perception. The histological studies highlighted that the ligation induces oedema and macrophagic infiltrate. Moreover, osmicated preparations revealed a decrease in axons’ compactness and diameter, together with a significant loss of myelin sheath. Repeated treatment with PNU‐282987 reduced the presence of oedema and macrophagic infiltrate and, on the coronal sections of the nerve, a significant higher myelin sheath, axonal diameter and number of fibers were observable. These results strongly suggest the pivotal role of alpha7 nAChR in the neuroprotection during neuropathy.

Analgesic and antineuropathic drugs acting through central cholinergic mechanisms.

The role of muscarinic and nicotinic cholinergic receptors in analgesia and neuropathic pain relief is relatively unknown. This review describes how such drugs induce analgesia or alleviate neuropathic pain by acting on the central cholinergic system. Several pharmacological strategies are discussed which increase synthesis and release of acetylcholine (ACh) from cholinergic neurons. The effects of their acute and chronic administration are described. The pharmacological strategies which facilitate the physiological functions of the cholinergic system without altering the normal modulation of cholinergic signals are highlighted. It is proposed that full agonists of muscarinic or nicotinic receptors should be avoided. Their activation is too intense and un-physiological because neuronal signals are distorted when these receptors are constantly activated. Good results can be achieved by using agents that are able to a) increase ACh synthesis, b) partially inhibit cholinesterase activity c) selectively block the autoreceptor or heteroreceptor feedback mechanisms. Activation of M1 subtype muscarinic receptors induces analgesia. Chronic stimulation of nicotinic (N1) receptors has neuronal protective effects. Recent experimental results indicate a relationship between repeated cholinergic stimulation and neurotrophic activation of the glial derived neurotrophic factor (GDNF) family. At least 9 patents covering novel chemicals for cholinergic system modulation and pain control are discussed.