Maria Pina Serra

Bortezomib-induced painful peripheral neuropathy: an electrophysiological, behavioral, morphological and mechanistic study in the mouse

Bortezomib is the first proteasome inhibitor with significant antineoplastic activity for the treatment of relapsed/refractory multiple myeloma as well as other hematological and solid neoplasms. Peripheral neurological complications manifesting with paresthesias, burning sensations, dysesthesias, numbness, sensory loss, reduced proprioception and vibratory sensitivity are among the major limiting side effects associated with bortezomib therapy. Although bortezomib-induced painful peripheral neuropathy is clinically easy to diagnose and reliable models are available, its pathophysiology remains partly unclear. In this study we used well-characterized immune-competent and immune-compromised mouse models of bortezomib-induced painful peripheral neuropathy. To characterize the drug-induced pathological changes in the peripheral nervous system, we examined the involvement of spinal cord neuronal function in the development of neuropathic pain and investigated the relevance of the immune response in painful peripheral neuropathy induced by bortezomib. We found that bortezomib treatment induced morphological changes in the spinal cord, dorsal roots, dorsal root ganglia (DRG) and peripheral nerves. Neurophysiological abnormalities and specific functional alterations in Aδ and C fibers were also observed in peripheral nerve fibers. Mice developed mechanical allodynia and functional abnormalities of wide dynamic range neurons in the dorsal horn of spinal cord. Bortezomib induced increased expression of the neuronal stress marker activating transcription factor-3 in most DRG. Moreover, the immunodeficient animals treated with bortezomib developed a painful peripheral neuropathy with the same features observed in the immunocompetent mice. In conclusion, this study extends the knowledge of the sites of damage induced in the nervous system by bortezomib administration. Moreover, a selective functional vulnerability of peripheral nerve fiber subpopulations was found as well as a change in the electrical activity of wide dynamic range neurons of dorsal horn of spinal cord. Finally, the immune response is not a key factor in the development of morphological and functional damage induced by bortezomib in the peripheral nervous system.

Tissue distribution of Ret, GFRalpha-1, GFRalpha-2 and GFRalpha-3 receptors in the human brainstem at fetal, neonatal and adult age

Occurrence and localization of receptor components of the glial cell line-derived neurotrophic factor (GDNF) family ligands, the Ret receptor tyrosine kinase and the GDNF family receptor (GFR) alpha-1 to -3, were examined by immunohistochemistry in the normal human brainstem at fetal, neonatal, and adult age. Immunoreactive elements were detectable at all examined ages with uneven distribution and consistent pattern for each receptor. As a rule, the GFRalpha-1 and GFRalpha-2 antisera produced the most abundant and diffuse tissue labelling. Immunoreactive perikarya were observed within sensory and motor nuclei of cranial nerves, dorsal column nuclei, olivary nuclear complex, reticular formation, pontine nuclei, locus caeruleus, raphe nuclei, substantia nigra, and quadrigeminal plate. Nerve fibers occurred within gracile and cuneate fasciculi, trigeminal spinal tract and nucleus, facial, trigeminal, vestibular and oculomotor nerves, solitary tract, medial longitudinal fasciculus, medial lemniscus, and inferior and superior cerebellar peduncles. Occasionally, glial cells were stained. Age changes were appreciable in the distribution pattern of each receptor. On the whole, in the grey matter, labelled perikarya were more frequently observed in pre- and perinatal than in adult specimens; on the other hand, in discrete regions, nerve fibers and terminals were abundant and showed a plexiform arrangement only in adult tissue; finally, distinct fiber systems in the white matter were immunolabelled only at pre- and perinatal ages. The results obtained suggest the involvement of Ret and GFRalpha receptors signalling in processes subserving both the organization of discrete brainstem neuronal systems during development and their functional activity and maintenance in adult life.

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.

Glial cell line-derived neurotrophic factor-like immunoreactivity in human trigeminal ganglion and nucleus

Glial cell line-derived neurotrophic factor (GDNF) is shown by immunohistochemistry in human trigeminal sensory system from 22 weeks of gestation to adulthood. In the trigeminal ganglion, a distinct subpopulation of GDNF-positive neurones is observed, which amounts to about 15% at early pre-term and adult ages and peaks to around 30% at perinatal ages. Labelled neurones are mostly small- and medium-sized. Occasionally, Schwann and satellite cells are stained. GDNF/substance P (SP) and GDNF/calcitonin gene-related peptide (CGRP) double stained neurones occur at all ages examined, whereas GDNF/trkA coexistence can be observed in pre- and full-term newborns only. Centrally, GDNF-immunostained fibers and terminal-like structures are mainly restricted to the spinal trigeminal nucleus, where they are codistributed with SP and CGRP. In the subnucleus caudalis, positive neurones can also be observed both in the superficial laminae and in the magnocellular part, with higher frequency in adults. These results suggest that GDNF may play a functional role in human trigeminal primary sensory neurones throughout life and provide indication for its possible involvement in the regulation of pain-related neuronal circuits in human trigeminal sensory system.

Ret, GFRalpha-1, GFRalpha-2 and GFRalpha-3 receptors in the human hippocampus and fascia dentata.

The immunohistochemical occurrence and localization of the receptor components of the glial cell line‐derived neurotrophic factor (GDNF) family ligands, the Ret receptor tyrosine kinase and GDNF family receptor (GFR) alpha‐1 to ‐3, is described in the human post‐mortem hippocampal formation at pre‐ and full‐term newborn, and adult age. Two different antibodies for each of the four‐receptor molecules were used. Western blot analysis indicates that the availability of GFRalpha receptor proteins may vary with age and post‐mortem delay. The immunohistochemical detectability of GFRalpha‐1, GFRalpha‐2, GFRalpha‐3 and Ret receptor molecules is shown in the rat up to 72 h post‐mortem. In the human specimens, labelled neuronal perikarya were detectable for each receptor protein at all examined ages, with prevalent localization in the pyramidal layer of the Ammons horn and hilus and granular layer of the fascia dentata. In the adult subjects, abundant punctate‐like structures were also present. Labelled glial elements were identifiable. Comparison of the pattern of immunoreactive elements among young and adult subjects suggests that the intracellular distribution of the GDNF family ligands may vary between pre‐ and perinatal life and adult age. The results obtained suggest the involvement of the Ret and GFRalpha receptors signalling in processes subserving both the organization of this cortical region during development and the functional activity and maintenance of the mature hippocampal neurons.

Polysialylated-neural cell adhesion molecule (PSA-NCAM) in the human trigeminal ganglion and brainstem at prenatal and adult ages

BackgroundThe polysialylated neuronal cell adhesion molecule (PSA-NCAM) is considered a marker of developing and migrating neurons and of synaptogenesis in the immature vertebrate nervous system. However, it persists in the mature normal brain in some regions which retain a capability for morphofunctional reorganization throughout life. With the aim of providing information relevant to the potential for dynamic changes of specific neuronal populations in man, this study analyses the immunohistochemical occurrence of PSA-NCAM in the human trigeminal ganglion (TG) and brainstem neuronal populations at prenatal and adult age.ResultsWestern blot analysis in human and rat hippocampus supports the specificity of the anti-PSA-NCAM antibody and the immunodetectability of the molecule in postmortem tissue. Immunohistochemical staining for PSA-NCAM occurs in TG and several brainstem regions during prenatal life and in adulthood. As a general rule, it appears as a surface staining suggestive of membrane labelling on neuronal perikarya and proximal processes, and as filamentous and dot-like elements in the neuropil. In the TG, PSA-NCAM is localized to neuronal perikarya, nerve fibres, pericellular networks, and satellite and Schwann cells; further, cytoplasmic perikaryal staining and positive pericellular fibre networks are detectable with higher frequency in adult than in newborn tissue. In the adult tissue, positive neurons are mostly small- and medium-sized, and amount to about 6% of the total ganglionic population. In the brainstem, PSA-NCAM is mainly distributed at the level of the medulla oblongata and pons and appears scarce in the mesencephalon. Immunoreactivity also occurs in discretely localized glial structures. At all ages examined, PSA-NCAM occurs in the spinal trigeminal nucleus, solitary nuclear complex, vestibular and cochlear nuclei, reticular formation nuclei, and most of the precerebellar nuclei. In specimens of different age, the distribution pattern remains fairly steady, whereas the density of immunoreactive structures and the staining intensity may change and are usually higher in newborn than in adult specimens.ConclusionThe results obtained show that, in man, the expression of PSA-NCAM in selective populations of central and peripheral neurons occurs not only during prenatal life, but also in adulthood. They support the concept of an involvement of this molecule in the structural and functional neural plasticity throughout life. In particular, the localization of PSA-NCAM in TG primary sensory neurons likely to be involved in the transmission of protopathic stimuli suggests the possible participation of this molecule in the processing of the relevant sensory neurotransmission.

GDNF family ligand receptor components Ret and GFRalpha-1 in the human trigeminal ganglion and sensory nuclei

The occurrence of Ret and GFRalpha-1 receptors is shown by immunohistochemistry in the human trigeminal sensory system at pre-, postnatal and adult age. Receptor-labeled neurons occur in both trigeminal ganglion and mesencephalic nucleus. In adult trigeminal ganglion, about 75% of Ret- and 65% of GFRalpha-1-labeled neurons are small- and medium-sized. The proportion of Ret+ and GFRalpha-1+ trigeminal ganglion neurons in the adult is about 25 and 60%, respectively. The majority of Ret+ are double labeled for GFRalpha-1 and glial cell line-derived neurotrophic factor (GDNF). Most of the GFRalpha-1+ cells contain GDNF and about 50% of them contain Ret. Triple labeling shows many Ret+/GDNF+/GFRalpha-1+ neurons, but also a number of Ret-/GDNF+/GFRalpha-1+ and Ret+/GDNF-/GFRalpha-1+ cells. Both Ret+ and GFRalpha-1+ neuronal subpopulations overlap with that containing calcitonin gene-related peptide. Ret+ pericellular basket-like nerve fibers occur in the adult trigeminal ganglion. Centrally, immunoreactivity is restricted to the spinal nucleus pars caudalis and pars interpolaris and to the mesencephalic nucleus. In adult specimens, Ret+ nerve fibers and puncta gather in the inner substantia gelatinosa. Ret+ neurons occur in the spinal nucleus and are more frequent in newborn than in adult subjects. Central GFRalpha-1+-labeled neurons and punctate elements are sparse. These findings support the involvement of GDNF and possibly other cognate ligands in the trophism of human trigeminal primary sensory neurons from prenatal life to adulthood, indicating a selective commitment to cells devoted to protopathic and proprioceptive sensory transmission. They also support the possibility that receptor molecules other than Ret could be active in transducing the ligand signal.

High affinity neurotrophin receptors in the human pre-term newborn, infant and adult cerebellum

The immunohistochemical occurrence of the high affinity neurotrophin (NT) receptors trkA, trkB, and trkC is shown in the pre‐term newborn, infant, and adult human post‐mortem cerebellum. Immunoreactive neuronal perikarya and processes were observed in all specimens examined, where they appeared unevenly distributed in the cerebellar cortical layers and deep nuclei, and showed regional differences among cerebellar lobules and folia. The trk receptor‐antibodies, tested by Western blot on human cerebellum homogenates, revealed multiple immunoreactive bands for trkA and single bands for trkB and trkC. The results obtained show the tissue localization of the trk receptor‐like immunoreactivity in the human cerebellum from prenatal to adult age. The analysis for codistribution of the receptors with the relevant ligand and among the receptors in discrete cortical and deep nuclei tissue fields shows a wide variety of conditions, from a good similarity in terms of type and density of labeled structures, to a lack of correspondence, and suggests the possibility of colocalization of trk receptors with the relevant neurotrophin and among them in the cerebellar cortex. These results sustain the concept that the neurotrophin trophic system participates in the development, differentiation, and maintenance of the human cerebellar connectivity and support the possibility of a multifactorial trophic support for the neurotrophins through target‐derived and local mechanisms.

Neurturin, persephin, and artemin in the human pre- and full-term newborn and adult hippocampus and fascia dentata.

The immunochemical occurrence and localization of the Glial cell line-derived neurotrophic factor (GDNF) family ligands neurturin (NTN), persephin (PSP), and artemin (ART) is described in the human postmortem hippocampus and fascia dentata from subjects aged 21 weeks of gestation to 88 years. The detectability of NTN, PSP, and ART is shown in the rat by Western blot and immunohistochemistry up to 70 h postmortem. In the human tissue, labeled neuronal perikarya were detectable for each trophin at all examined ages, with prevalent localization in the pyramidal layer of the Ammons horn and hilus and granular layer of the fascia dentata. In the adult subjects, punctate elements were also present. Comparison of the pattern of immunoreactive structures among young and adult subjects suggests that intracellular distribution and/or trafficking of the GDNF family ligands may undergo age-related changes. Labeled glial elements were also identifiable. Western blot analysis indicates that the availability of the dimeric and monomeric forms of the trophins may vary with age and postmortem delay. The results obtained suggest the involvement of NTN, PSP, and ART in processes subserving both the organization of this cortical region during development and the functional activity and maintenance of the mature human hippocampal neurons.

Neurotrophin-like immunoreactivity in the human pre-term newborn, infant, and adult cerebellum

The immunohistochemical occurrence of the neurotrophin (NT) proteins nerve growth factor (NGF), brain‐derived neurotrophic factor (BDNF), neurotrophin‐4 (NT‐4), and neurotrophin‐3 (NT‐3) is shown in the pre‐term newborn, infant, and adult human post‐mortem cerebellum. The NT‐like immunoreactive structures were unevenly distributed and showed regional differences among cerebellar lobules and folia. NGF‐, NT‐4‐, and NT‐3‐positive neuronal perikarya were observed in all specimens examined. At variance with the other neurotrophins, the BDNF antiserum labelled neuronal cell bodies only in newborn life and infancy, as well as extensive nerve fibre systems, whose density increased with age. The NT‐antibodies, tested by Western blot on human cerebellum homogenates, revealed immunoreactive bands corresponding to proteins of heterogenous molecular weight. The results obtained provide a first demonstration of the tissue localization of the NTs in the human cerebellum from perinatal to adult age, thus suggesting their involvement in the development, differentiation and maintenance of the cerebellar connectivity. Codistribution of the four NTs or sets of them was observed in cortical and deep nuclei neurons. Multiple trophic roles for NTs, encompassing the classic target‐derived and local mechanisms of support, are envisaged as significant in development, differentiation, and maintenance of the human cerebellar connectivity.