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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., Italian Journal of Anatomy and Embryology, Vol 119, No 1 (Supplement) 2014

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., Italian Journal of Anatomy and Embryology, Vol 119, No 1 (Supplement) 2014

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., Italian Journal of Anatomy and Embryology, Vol 119, No 1 (Supplement) 2014

Bortezomib (BTZ), a selective proteasome inhibitor, is an antitumor drug used to treat multiple myeloma. As a side effect, BTZ determines a painful peripheral neuropathy (PPN), often refractory to management. Chronic BTZ administration in female Wistar rats induced a PPN in which the development of mechanical allodynia is associated to an increase in the expression of TRPV1 and CGRP in the dorsal root ganglia (DRG) and spinal cord dorsal horn. In this study we examine the possible role of two standard analgesics, Gabapentin (Gaba) and buprenorfin (Bupre), in modulating the expression of TRPV1 and CGRP in the DRG and spinal cord of rats chronically treated with BTZ. To this aim, female Wistar rats were treated with BTZ 0.20 mg/kg, 3 times a week for 8 weeks (i.v.). Then Gaba (100 mg/kg, daily, p.o.) and Bupre (28,8 µg/kg, daily, s.c.) were orally administered in a curative schedule for 2 weeks. The expression of TRPV1 and CGRP, a neuropeptide involved in nociceptive neurotransmission, was examined in L4-L5 DRG and spinal cord segments by means of western blot (WB) and immunohistochemistry. WB analysis did not show any statistically significant change of protein levels in DRG and spinal cord. Instead, chronic BTZ treatment followed by a 2 weeks follow-up period affected TRPV1 expression by inducing an increase in the proportion of TRPV1-like immunoreactive (LI) DRG neurons, whereas did not affect the percentage of CGRP-LI neurons. Labeled perikarya were mostly of small- and medium-size. No BTZ-induced changes in immunolabeling were appreciable in the dorsal horn of the spinal cord. Treatment with Gaba and Bupre reduced levels of TRPV1 protein in spinal cord homogenates, whereas had no effect on DRG protein levels. None of the analgesics appeared to reverse in a statistically significant way the BTZ-induced increase of labelled DRG neurons nor induced any change in the detectability of positive innervation in the spinal cord dorsal horn.

Bortezomib (BTZ), a selective proteasome inhibitor, is a potent antineoplastic drug to treat multiple myeloma. In clinical practice, the most significant side effect of its administration, is a peripheral painful sensory neuropathy (PN), characterized by numbness and tingling in a distal stocking-glove distribution as well as distal paresthesia and generally associated with impairment of Adelta and C type primary afferent fibers. At the moment the therapies for PN are not really effective and almost only symptomatic. To evaluate if BTZ-induced sensory deficits are associated to loss of epidermal nerve fibers as is observed in other PN conditions, here we examine the effect of a well-established chronic BTZ-treatment schedule on the number of intraepidermal nerve fibers (IENF) in a rat model of BTZ-induced PN and compared the outcome of chronic BTZ-treatment followed by two-weeks of follow up with that of animals that received a 2-weeks treatment period with two analgesic drugs commonly used in clinical practice such as Gabapentin (Gaba) and buprenorphine (Bupre). To these aims, female Wistar rats were treated with BTZ 0.20 mg/kg, 3 times a week for 8 weeks (i.v.). Then analgesic were orally administered according to the following curative schedule: Gaba (100 mg/kg, daily, p.o.), Bupre (28,8 μg/kg, daily, s.c.). At the end of treatments, the hindlimb footpad skin was examined by immunohistochemistry for the pan-neuronal marker PGP9.5 and the neuropeptide calcitonin gene-related peptide (CGRP), a neuropeptide involved in pain perception and neurotransmission. Labelled nerve fibers were distributed in the reticular and papillary dermis and within the epidermal lining. Quantitative evaluation of IENF showed that, according to the painful symptoms induced by BTZ, the mean linear density of CGRP-labelled IENF normalized by that of the PGP9.5-labelled ones shows a statistically significant decrease in BTZ-treated rats. None of the analgesics used in this study appeared to reverse the BTZ-induced loss of IENF. Results obtained suggest that the CGRP-positive innervation is likely involved in the persistence of BTZ-induced pain and that detectability of CGRP-positive IENF appears not to be related to the effectiveness of the selected analgesics.

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. Carozzi VA, Renn CL, Bardini M. et al. PLoS One. 2013; 8(9):e72995.

The skin is equipped with specialized mechanoreceptors that allow the perception of the slightest brush. Indeed, some mechanoreceptors can detect even nanometer-scale movements. Movement is transformed into electrical signals via the gating of mechanically activated ion channels at sensory endings in the skin. The sensitivity of Piezo mechanically gated ion channels is controlled by stomatin-like protein-3 (STOML3), which is required for normal mechanoreceptor function. Here we identify small-molecule inhibitors of STOML3 oligomerization that reversibly reduce the sensitivity of mechanically gated currents in sensory neurons and silence mechanoreceptors $\textit{in vivo}$. STOML3 inhibitors in the skin also reversibly attenuate fine touch perception in normal mice. Under pathophysiological conditions following nerve injury or diabetic neuropathy, the slightest touch can produce pain, and here STOML3 inhibitors can reverse mechanical hypersensitivity. Thus, small molecules applied locally to the skin can be used to modulate touch and may represent peripherally available drugs to treat tactile-driven pain following neuropathy.

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.