Cristina Picci

Bortezomib Treatment Produces Nocifensive Behavior and Changes in the Expression of TRPV1, CGRP, and Substance P in the Rat DRG, Spinal Cord, and Sciatic Nerve

To investigate neurochemical changes associated with bortezomib-induced painful peripheral neuropathy (PN), we examined the effects of a single-dose intravenous administration of bortezomib and a well-established “chronic” schedule in a rat model of bortezomib-induced PN. The TRPV1 channel and sensory neuropeptides CGRP and substance P (SP) were studied in L4-L5 dorsal root ganglia (DRGs), spinal cord, and sciatic nerve. Behavioral measures, performed at the end of the chronic bortezomib treatment, confirmed a reduction of mechanical nociceptive threshold, whereas no difference occurred in thermal withdrawal latency. Western blot analysis showed a relative increase of TRPV1 in DRG and spinal cord after both acute and chronic bortezomib administration. Reverse transcriptase-polymerase chain reaction revealed a decrease of TRPV1 and CGRP mRNA relative levels after chronic treatment. Immunohistochemistry showed that in the DRGs, TRPV1-, CGRP-, and SP-immunoreactive neurons were mostly small- and medium-sized and the proportion of TRPV1- and CGRP-labeled neurons increased after treatment. A bortezomib-induced increase in density of TRPV1- and CGRP-immunoreactive innervation in the dorsal horn was also observed. Our findings show that bortezomib-treatment selectively affects subsets of DRG neurons likely involved in the processing of nociceptive stimuli and that neurochemical changes may contribute to development and persistence of pain in bortezomib-induced PN.

TRPV1 receptor in the human trigeminal ganglion and spinal nucleus: immunohistochemical localization and comparison with the neuropeptides CGRP and SP.

This work presents new data concerning the immunohistochemical occurrence of the transient receptor potential vanilloid type‐1 (TRPV1) receptor in the human trigeminal ganglion (TG) and spinal nucleus of subjects at different ontogenetic stages, from prenatal life to postnatal old age. Comparisons are made with the sensory neuropeptides calcitonin gene‐related peptide (CGRP) and substance P (SP). TRPV1‐like immunoreactive (LI) material was detected by western blot in homogenates of TG and medulla oblongata of subjects at prenatal and adult stages of life. Immunohistochemistry showed that expression of the TRPV1 receptor is mostly restricted to the small‐ and medium‐sized TG neurons and to the caudal subdivision of the spinal trigeminal nucleus (Sp5C). The extent of the TRPV1‐LI TG neuronal subpopulation was greater in subjects at early perinatal age than at late perinatal age and in postnatal life. Centrally, the TRPV1 receptor localized to fibre tracts and punctate elements, which were mainly distributed in the spinal tract, lamina I and inner lamina II of the Sp5C, whereas stained cells were rare. The TRPV1 receptor colocalized partially with CGRP and SP in the TG, and was incompletely codistributed with both neuropeptides in the spinal tract and in the superficial laminae of the Sp5C. Substantial differences were noted with respect to the distribution of the TRPV1‐LI structures described in the rat Sp5C and with respect to the temporal expression of the receptor during the development of the rat spinal dorsal horn. The distinctive localization of TRPV1‐LI material supports the concept of the involvement of TRPV1 receptor in the functional activity of the protopathic compartment of the human trigeminal sensory system, i.e. the processing and neurotransmission of thermal and pain stimuli.

The human cuneate nucleus contains discrete subregions whose neurochemical features match those of the relay nuclei for nociceptive information

The present paper is aimed at defining distinctive subdivisions of the human cuneate nucleus (Cu), evident from prenatal to old life, whose occurrence has never been clearly formalized in the human brain, or described in other species so far. It extends our early observations on the presence of gray matter areas that host strong substance P (SP) immunoreactivity in the territory of the human Cu and adjacent cuneate fascicle. Here we provide a three-dimensional reconstruction of the Cu fields rich in SP and further identify those areas by means of their immunoreactivity to the neuropeptides SP, calcitonin gene-related peptide, methionine- and leucine-enkephalin, peptide histidine-isoleucine, somatostatin and galanin, to the trophins glial cell line-derived neurotrophic factor and brain-derived neurotrophic factor, and to the neuroplasticity proteins polysialylated neural cell adhesion molecule and growth-associated protein-43. The presence, density and distribution of immunoreactivity for each of these molecules closely resemble those occurring in the superficial layers of the caudal spinal trigeminal nucleus (Sp5C). Myelin and Nissl stainings suggest that those Cu subregions and the Sp5C superficial layers share a similar histological aspect. This work establishes the existence of definite subregions, localized within the Cu territory, that bear the neurochemical and histological features of sensory nuclei committed to the neurotransmission of protopathic stimuli, including pain. These findings appear of particular interest when considering that functional, preclinical and clinical studies show that the dorsal column nuclei, classical relay station of fine somatic tactile and proprioceptive sensory stimuli, are also involved in pain neurotransmission.

Effect of resveratrol on plasmatic molecular indicators of brain tissue response to the hypoperfusion/ reperfusion challenge

It is well-documented that endocannabinoids (eCBs) and congeners show a neuroprotective role in several experimental models of brain injury and that changes in eCB levels in peripheral blood cells may reflect the severity of neurological insult. We have previously shown that the preventive administration of dietary natural compounds may increase the plasmatic levels of palmytoylethanolamide (PEA) and oleoylethanolamide (OEA) following the transient bilateral common carotid artery occlusion (BCCAO)-induced brain tissue challenge (1). Resveratrol (RVT), (3,4’, 5-trihidroxystilbene) is a strong natural antioxidant of polyphenolic structure found in grapes and red wine, with many physiological effects, including the prevention of lipid peroxidation in human LDL, inhibition of arachidonic acid metabolism, and platelet activity. RVT has been further shown to protect cerebral tissue and cardiac muscle from tissue damage caused by oxidative stress triggered by reperfusion (2) and has been proposed as a potential neuroprotective agent in treating acute states in focal cerebral ischemia injury (3). In this line, we intend to evaluate whether exogenous administration of RVT prior to induction of BCCAO followed by reperfusion influences the molecular changes occurring in cerebral cortex and plasma, with particular focus on the eCB system. With this aim, cerebral hypoperfusion was produced by a 30 min BCCAO followed by 60 min reperfusion (BCCAO/R). Animals were starved for 12 hours before surgery and 6 hours prior to ischemia RVT (40 mg/kg/0.45 ml of sunflower oil as vehicle) was administered via gavage. Biological samples of plasma, cerebrospinal fluid (CSF), and brain tissue were examined by HPLC, gel zymography, western blot and immunohistochemistry. Data obtained indicate that RVT appears to influence the outcome of BCCAO/R cerebral injury by modulating changes in levels of lipid hydroperoxides, markers of oxidative stress, eCBs and eCB congeners, expression of CB1 and CB2 receptors, peroxisome proliferator-activated receptor- (PPAR) alpha, ciclooxygenase-2 (COX-2) protein levels and enzymatic activity of matrix-metalloproteinase- 9 (MMP-9). Interestingly, changes in brain of some of these parameters, like lipid hydroperoxides, were also found in plasma. Results obtained suggest that exogenous administration of RVT may modulate the brain tissue compensatory or repair mechanisms triggered by the hypoperfusion/reperfusion and support the possible use of this molecule as treatment to prevent the BCCAO/R-induced brain insult. In addition, the finding that changes in plasma mirrored those found in cerebral tissue, opens to the possibility to test whether RSV exerts its positive activities in humans.

Dietary essential oil components in the prevention of hypoperfusion/reperfusion-induced tissue damage in the rat cerebral cortex

To extend our previous observations on the beneficial effect of dietary Pistacia lentiscus L. essential oil during cerebral bilateral common carotid artery occlusioninduced injury, we evaluated the activity of one of its major components, beta-caryophyllene (BCP), already known to possess peculiar biological activities, in Wistar rat cerebral cortex. Cerebral hypoperfusion was produced by a 30 min bilateral common carotid artery occlusion followed by 60 min reperfusion (BCCAO/R). Animals were starved for 12 hours before surgery and, 6 hours prior to hypoperfusion, BCP (40 mg/kg/0, 45 ml of sunflower oil as vehicle) was administered via gavage. Biological samples of brain tissue, plasma and cerebrospinal fluid (CSF) were examined by HPLC, western blot, gel zymography and immunohistochemistry and analyzed for fatty acids, expression of the enzyme ciclooxygenase-2 (COX-2), CB receptors for endocannabinoids (eCBs), and peroxisome proliferator-activated receptor (PPAR)-alpha and enzymatic activity of matrix-metalloprotease-9 (MMP9). Data obtained indicate that BCP appears to influence the outcome of BCCAO/R cerebral injury by modulating changes in levels of polyunsaturated fatty acids, biosynthesis of eCBs and eCB congeners, expression of CB1 and CB2 receptors, COX-2 protein levels and enzymatic activity of MMP9. Brain tissue response to the hypoperfusion/reperfusion-induced cerebral insult is modulated by dietary administration of BCP, suggesting the possible use of this molecule as nutritional treatment in neuroprevention. Work funded by Fondazione Banco di Sardegna.

Locus K: cuneate subnuclear regions in human dorsal column nuclei with neurochemical, cyto- and myeloarchitectural features of protopathic sensory nuclei

This study is aimed to further characterize on a neurochemical, histological and morphometric ground the human Locus K, a newly identified region in the human nucleus cuneatus that shares neurochemical features with protopathic second order sensory nuclei (Del Fiacco et al., 2013; Serra et al., 2013). Human brainstem sections were observed by means of ABC immunohistochemistry for calcitonin gene-related peptide (CGRP), substance P (SP) and transient receptor potential vanilloid type-1 receptor (TRPV1), Kluver-Barrera, Black Gold kit II and Nissl staining followed by computerized analysis of cell size and density. As for CGRP and SP, at both pre- and postnatal age, immunoreactivity to TRPV1 occurs in Locus K with a distribution alike that present in the spinal trigeminal nucleus, caudal part. Morphometric analysis shows that, in adult tissue, the mean diameter and density of Nissl stained neurons in the Locus K are consistent with those of the caudal spinal trigeminal and solitary nuclei, and different from those in the gracile, cuneate and external cuneate nuclei. Kluver-Barrera and Black Gold kit II staining shows that myelinated fibres, abundant in the main cuneate, gracile and trigeminal magnocellular nuclei, are scarce in both the Locus K and trigeminal substantia gelatinosa. Immunohistochemical and cyto- and myeloarchitectural analysis uphold the parallel neurochemical and structural arrangement for Locus K and protopathic nuclei in the human medulla oblongata, and support the concept that Locus K represents a special component of the human dorsal column nuclei. Work funded by Fondazione Banco di Sardegna.

The rat dorsal column nuclei contain a region homologous to the human Locus K

Locus K is a newly identified region within the territory of the human nucleus cuneatus that shares neurochemical and histological features with protopathic second order sensory nuclei (Del Fiacco et al., 2013; Serra et al., 2013; SIAI 2014). This work is aimed at examining the rat dorsal column nuclei in order to ascertain whether a structure homologous to the human Locus K occurs in the rat brain. Rat brainstem sections were observed by means of ABC and fluorescence immunohistochemistry for neuropeptides calcitonin gene-related peptide (CGRP) and substance P (SP), Kluver-Barrera and Nissl staining. Results of the peptide immunoreactive structures in the rat dorsal column nuclei are in general good agreement with findings in previous studies (Hoeflinger et al., 1993). However, at caudalmost levels of the complex, in the territory of the cuneate fascicle and dorsal to the caudal pole of the cuneate nucleus, a small column of gray matter area can be identified that contains a dense plexus of varicose labelled nerve fibres. The observed discrete region has never distinctly described beforehand. Both its position and aspect at neuropeptide-immunoreactivity resemble those of the Locus K we detected in the human dorsal column nuclei, allowing the possibility that it represents the its homologous nucleus in the rat brain. Work funded by Fondazione Banco di Sardegna.

Locus K: a novel territory of the human dorsal column nuclei

Bortezomib (BTZ) is a proteasome inhibitor used as first-line therapy for multiple myeloma. However, its administration induces the development of severe painful peripheral neuropathy (PPN). This painful condition is an important medical need since the available treatments are actually ineffective. We recently described a mice model of PPN that shares most of the conditions found in patients treated chronically with BTZ (Carozzi et al., 2013). In fact, BTZ determines dysfunction of all fiber types in sensory nerves and, at least in mice, alters the electrical activity of the spinal dorsal horn neurons. This alteration of the basal electrophysiological activity induces also relevant changes in the central nociceptive transmission. In this work we characterize the neuroprotective effects of an imidazoline receptor 2 ligand (CR4056) able to allosterically inhibit the activity of monoamine oxidase-A, a key enzyme in the regulation of neuropathic pain. Wistar rats were treated with BTZ 0.20 mg/kg, 3 times a week for 8 weeks (i.v). Then CR4056 was orally administered in a curative schedule at 6 mg/kg, once a day, for 2 weeks. Gabapentin (100 mg/kg, daily, p.o.) and buprenorphine (28,8μg/kg, daily, s.c.) were used as internal analgesic standards. At the end of both BTZ and analgesic treatments, we measured the caudal and sciatic nerve conduction velocity (NCV), the morphological/morphometrical alterations in the caudal nerve and the neuropathic pain development. BTZ treatment induced a significant impairment of sensory, but not motor NCV, slight hyperalgesia, significant mechanical allodynia and clearing of myelinated fibers in the caudal nerves. After two weeks of follow up animals did not spontaneously recover functional, morphological and behavioral abnormalities while the 2 weeks-treatment with CR4056 (but not with gabapentine and buprenorphine) significantly resolved BTZ-induced mechanical allodynia. Results obtained show that CR4056 produces a marked analgesic effects against BTZ-induced neuropathic pain without signs of tolerance.

Imidazoline Receptor 2 is an Effective Target for Neuropathic Pain in a Murine Model of Bortezomib-Induced Peripheral Neuropathy

Bortezomib (BTZ) is a proteasome inhibitor used as first-line therapy for multiple myeloma. However, its administration induces the development of severe painful peripheral neuropathy (PPN). This painful condition is an important medical need since the available treatments are actually ineffective. We recently described a mice model of PPN that shares most of the conditions found in patients treated chronically with BTZ (Carozzi et al., 2013). In fact, BTZ determines dysfunction of all fiber types in sensory nerves and, at least in mice, alters the electrical activity of the spinal dorsal horn neurons. This alteration of the basal electrophysiological activity induces also relevant changes in the central nociceptive transmission. In this work we characterize the neuroprotective effects of an imidazoline receptor 2 ligand (CR4056) able to allosterically inhibit the activity of monoamine oxidase-A, a key enzyme in the regulation of neuropathic pain. Wistar rats were treated with BTZ 0.20 mg/kg, 3 times a week for 8 weeks (i.v). Then CR4056 was orally administered in a curative schedule at 6 mg/kg, once a day, for 2 weeks. Gabapentin (100 mg/kg, daily, p.o.) and buprenorphine (28,8μg/kg, daily, s.c.) were used as internal analgesic standards. At the end of both BTZ and analgesic treatments, we measured the caudal and sciatic nerve conduction velocity (NCV), the morphological/morphometrical alterations in the caudal nerve and the neuropathic pain development. BTZ treatment induced a significant impairment of sensory, but not motor NCV, slight hyperalgesia, significant mechanical allodynia and clearing of myelinated fibers in the caudal nerves. After two weeks of follow up animals did not spontaneously recover functional, morphological and behavioral abnormalities while the 2 weeks-treatment with CR4056 (but not with gabapentine and buprenorphine) significantly resolved BTZ-induced mechanical allodynia. Results obtained show that CR4056 produces a marked analgesic effects against BTZ-induced neuropathic pain without signs of tolerance.

Dietary essential oil components in the prevention of ischemia/reperfusion-induced tissue damage in the cerebral cortex

Bortezomib (BTZ) is a proteasome inhibitor used as first-line therapy for multiple myeloma. However, its administration induces the development of severe painful peripheral neuropathy (PPN). This painful condition is an important medical need since the available treatments are actually ineffective. We recently described a mice model of PPN that shares most of the conditions found in patients treated chronically with BTZ (Carozzi et al., 2013). In fact, BTZ determines dysfunction of all fiber types in sensory nerves and, at least in mice, alters the electrical activity of the spinal dorsal horn neurons. This alteration of the basal electrophysiological activity induces also relevant changes in the central nociceptive transmission. In this work we characterize the neuroprotective effects of an imidazoline receptor 2 ligand (CR4056) able to allosterically inhibit the activity of monoamine oxidase-A, a key enzyme in the regulation of neuropathic pain. Wistar rats were treated with BTZ 0.20 mg/kg, 3 times a week for 8 weeks (i.v). Then CR4056 was orally administered in a curative schedule at 6 mg/kg, once a day, for 2 weeks. Gabapentin (100 mg/kg, daily, p.o.) and buprenorphine (28,8μg/kg, daily, s.c.) were used as internal analgesic standards. At the end of both BTZ and analgesic treatments, we measured the caudal and sciatic nerve conduction velocity (NCV), the morphological/morphometrical alterations in the caudal nerve and the neuropathic pain development. BTZ treatment induced a significant impairment of sensory, but not motor NCV, slight hyperalgesia, significant mechanical allodynia and clearing of myelinated fibers in the caudal nerves. After two weeks of follow up animals did not spontaneously recover functional, morphological and behavioral abnormalities while the 2 weeks-treatment with CR4056 (but not with gabapentine and buprenorphine) significantly resolved BTZ-induced mechanical allodynia. Results obtained show that CR4056 produces a marked analgesic effects against BTZ-induced neuropathic pain without signs of tolerance.