DIPARTIMENTO DI BIOLOGIA
CORSO DI LAUREA IN BIOLOGIA APPLICATA ALLA BIOMEDICINA
CURRICULUM FISIOPATOLOGICO
“Preclinical evaluation of NGF-based therapies on a
mouse model of osteoarthritis and diabetes”
ANNO ACCADEMICO 2015/2016
SESSIONE MAGGIO 2017
RELATORI: LAUREANDA:
Cattaneo Antonino Fasano Elena
INTRODUCTION ... 4
1. NERVE GROWTH FACTOR: A NEW EUREKA IN NEUROSCIENCE ... 4
1.1 NEUROTROPHINS AND THEIR RECEPTORS ... 5
1.2 ROLE OF NGF IN THE DORSAL ROOT GANGLION (DRG) ... 11
1.4 ROLE OF NGF IN BONE METABOLISM ... 20
1.5 OSTEOARTHRITIS ... 21
1.5.1NEWTHERAPIESFOROA ... 23
1.5.1.1 αD11 ... 26
1.5.1.2 MNAC13 ... 30
1.6PAINFULDIABETICNEUROPATHY ... 34
1.6.1 TREATMENT OF PDN WITH NGF ... 38
AIMS OF THE RESEARCH THESIS ... 40
MATERIAL AND METHODS ... 42
3.1OSTEOARTHRITIS ... 42 3.1.1 ANIMALS ... 42 3.1.2 INDUCTION OF OSTEOARTHRITIS ... 42 3.1.3 ADMINISTRATION OF ANTIBODIES ... 42 3.1.4 INCAPACITANCE TESTER ... 43 3.1.5 KNEE HISTOLOGY ... 44 3.2DIABETES ... 48 3.2.1 ANIMALS ... 48 3.2.2 ADMINISTRATION OF pNGF ... 48 3.2.3 MECHANICAL SENSITIVITY ... 49 RESULTS ... 51
4.1EFFECTSOFASINGLEDOSEOFMNAC13OR ΑD11ANTIBODIESBEFOREOA
INDUCTION ... 51
4.1.1. WEIGHT BEARING TEST ... 51
4.2.EFFECTSOFASINGLEDOSEOFMNAC13OR ΑD11ANTIBODIESAFTEROA INDUCTION ... 54
4.2.1. WEIGHT BEARING TEST ... 54
4.3EFFECTSOFAPROLONGEDADMINISTRATIONOFMNAC13 ... 56
4.3.1 WEIGHT BEARING TEST ... 56
.4.3.2. KNEES HISTOLOGY ... 57
PART 2: EFFECT OF ADMINISTRATION OF PNGF IN DB/DB MICE ... 61
DISCUSSION ... 63
5.1 OSTEOARTHRITIS ... 63
5.2 DIABETES ... 67
INTRODUCTION
1. NERVE GROWTH FACTOR: A NEW EUREKA IN NEUROSCIENCE
The first cell growth factor, nerve growth factor (NGF), was discovered by Rita Levi-Montalcini in the early 1950s. At the end of the nineteenth century, Santiago Ramon y Cajal had envisaged that life at the neuronal level requires trophic support. The proof was obtained by work initiated by Rita Levi-Montalcini in 1949 in Victor Hamburger's Department of Zoology in St. Louis. In brief, her unpredictable laboratory protocols included (i) the transplantation of mouse sarcoma 180 into chicken embryo, leading to the growth of sympathetic and sensory nerves, (ii) snake venom used to destroy DNA in sarcoma homogenate, leading to greater nerve growth than that induced by a sarcoma itself and (iii) the homogenate of male mouse submandibular glands (the mammalian homologue of snake venom), leading to even greater nerve growth than with snake venom. This cascade of nerve growth findings was marked by a rare combination of scientific reasoning, intuition and chance, the latter ‘favours only the prepared mind’ (Louis Pasteur).4 The yet unknown molecule mediating such a heuristic cascade was initially named nerve growth-stimulating factor, later NGF 1,2,3. In attempt to purify the
tumour-derived factor, Levi-Montalcini and Stanley Cohen used snake venom as a rich source of phosphodiesterase, a nucleic acid-destroying enzyme, for the separation of nucleic acids and protein fractions in the tumour material. To their great surprise, the tumour fraction containing the snake venom was several 1000-fold more potent both in vitro and in vivo than control tumour homogenate in promoting nerve growth. Further on the road of discovery, Levi-Montalcini and Cohen examined the mammalian homologue of the snake venom, the salivary gland and found that the male mouse submandibular glands are an even richer source of the same nerve growth- stimulating
activity found in both tumour and snake venom. Thus, male mouse submandibular glands appeared to be a new and possibly largest source of NGF, making it possible to isolate and purify consistent amounts of this molecule.4
1.1 NEUROTROPHINS AND THEIR RECEPTORS
Nerve growth factor belongs to a family of proteins called neurotrophins. It was purified as a survival factor for cultured sympathetic and sensory neurons from the submaxillary glands of male mice, where it is expressed at extraordinarily high levels, greatly simplifying its purification5,6. It was shown that NGF is synthesized and secreted by
sympathetic and sensory target organs, and after secretion, it is captured through receptor interactions in nerve terminals, where it acts locally to regulate target innervation and nerve terminal function7,8. After internalization, NGF is transported to
neuronal cell bodies, where it acts to promote neuronal survival and differentiation9.
Extending the observations on NGF, each of the other neurotrophins has also been shown to be a target-derived neurotrophic factor10,11. Within targets, expression of these
proteins is localized to specialized end organs, such as hair follicles, that are innervated by the axons of responsive neurons. Through receptor interactions, internalization and retrograde transport, each of these proteins has local actions affecting growth cone behaviour and synaptic function, as well as actions at a distance in the cell body and nucleus.30
Neurotrophins are essential for the development of the vertebrate nervous system and for neuronal cells survival. In addition to this, they can also regulate axonal and dendritic growth and guidance, synaptic structure and connections, neurotransmitter release, long-term potentiation (LTP) and synaptic plasticity12. For example,
neurotrophins and their receptor influence many aspects of neuronal activity that result in the generation of new synaptic connections, which can be long lasting.13 Alterations
in neurotrophin levels have profound effects on a wide variety of phenomena, including myelination, regeneration, pain, aggression, depression and substance abuse.25
The actions of neurotrophins depend on two different transmembrane-receptor signalling systems14― the Trk receptor tyrosine kinases and the p75 neurotrophin
receptor.15,16 Despite considerable progress in understanding the roles of these receptors,
additional mechanism are needed to explain the many cellular and synaptic interactions that occur between neurons. An emerging view is that neurotrophin receptors act as sensors for various extracellular and intracellular inputs, and several new mechanisms have recently been put forward.
It is well established that the overall levels of neurotrophins determine the balance between cell survival and apoptosis during development. Neural activity has profound effects on the levels of neurotrophins. Indeed, the idea that neurotrophins are crucial for synaptic plasticity came from observations that they are synthesized and released in an activity-dependent manner.17,18,19 NGF and brain-derived neurotrophic factor (BDNF)
messenger RNAs are highly regulated by electrical stimulation and epileptic activity20,
and BDNF in particular is rapidly released by neuronal activity during periods of activity-dependent synaptic remodelling.21,5,22,23
Studies of mice that express reduced levels of neurotrophins have shown surprising effects on adult brain function and behaviour. Mice that completely lack neurotrophins die during the first few weeks following birth. Heterozygous mice in which
neurotrophin levels are reduced by half are viable but, strikingly, they show other deficits, for example in memory acquisition and retention.24,25
The neurotrophins and their genes share homologies in sequence and structure. The organization of the genomic segments adjacent to these genes is also similar. Together, these observations provide compelling evidence that the neurotrophin genes have arisen through successive duplications of a portion of the genome derived from an ancestral chordate26. Their genes share many similarities, including the existence of multiple
promoters. The protein product of each gene includes a signal sequence and a prodomain, followed by the mature neurotrophin sequence. Thus, each gene product must be processed by proteolysis to form a mature protein. Indeed, the neurotrophins are initially synthesized as precursor or pro-neurotrophins, which are cleaved to produce the mature proteins27 in the trans-Golgi network28. Lee in 2001 has demonstrated that
regulation of their maturation is an important post-transcriptional control point that limits and adds specificity to their actions29. Pro-neurotrophins are clevead
intracellularly by furin or pro-convertases at a highly conserved dibasic amino-acid cleveage site to release carboxy-terminal mature proteins. The mature proteins, which are about 12kDa in size, from stable, non-covalent dimers, and are normally expressed at very low levels during development. The amino-terminal half (or pro-domain) of the pro-neurotrophin is believed to be important for the proper folding and intracellular sorting of neurotrophins.25 The mature neurotrophin proteins are non-covalently associated homodimers. Although some neurotrophin monomers are able to form heterodimers with other neurotrophin monomers in vitro, there is no evidence that these heterodimers exist at significant concentrations in vivo30.
As already said, the neurotrophins interact with two entirely distinct classes of receptors30. The first receptor to be discovered, named p75 neurotrophin receptor
(p75NTR), was identified as a low-affinity receptor for NGF, but was subsequently shown to bind each of the neurotrophins with a similar affinity31,32. p75NTR is a
member of the tumour necrosis receptor superfamily with an extracellular domain that includes four cysteine-rich motifs, a single transmembrane domain and a cytoplasmic domain that includes a ‘death’ domain similar to those present in other members of this family33,34.
Although this receptor does not contain a catalytic motif, it interacts with several proteins that transmit signals important for regulating neuronal survival and differentiation as well as synaptic plasticity.30 The three-dimensional structure of the
extracellular domain of p75NTR in association with an NGF dimer has demonstrated that each of the four cysteine-rich repeats participates in binding to NGF34.
Interestingly, p75NTR binds NGF along the interface between the two NGF monomers and binding results in a conformational change in NGF that alters the monomeric interface on the opposite side of the NGF dimer, eliminating the potential for binding of one NGF dimer to two p75NTR monomers. The results suggest that binding of NGF to p75NTR may result in dissociation of p75NTR multimers and are compatible with the possibility that tropomyosinrelated kinase (Trk) and p75NTR monomers simultaneously bind the same neurotrophin dimer.30 Of recent interest, a gene related to p75NTR,
named NRH-2, has recently been identified. The product of this gene lacks the extracellular cysteine-rich repeats present in p75NTR and fails to bind NGF, but it is able to interact and influence the ligand-binding properties of TrkA35.
In mammals, the three members of the Trk subfamily of receptor tyrosine kinases constitute the second major class of neurotrophin receptors25. The extracellular domain
of each of the Trk receptors consists of a cysteine-rich cluster followed by three leucine-rich repeats, another cysteine-leucine-rich cluster and two immunoglobulin-like domains. Each receptor spans the membrane once and is terminated with a cytoplasmic domain consisting of a tyrosine kinase domain surrounded by several tyrosines that serve as phosphorylation-dependent docking sites for cytoplasmic adaptors and enzymes. In contrast to interactions with p75NTR, the neurotrophins dimerize the Trk receptors, resulting in activation through transphosphorylation of the kinases present in their cytoplasmic domains. The four neurotrophins exhibit specificity in their interactions with the three members of this receptor family with NGF activating TrkA, BDNF and NT-4 activating TrkB, and NT-3 activating TrkC. In addition, NT-3 can activate the other Trk receptors with less efficiency. (Figure 1)
Figure 1 - Neurotrophin binding results in dimerization of each receptor. Neurotrophins bind selectively to specific Trk receptor, whereas all neurotrophins bind to p75. Trk receptors contain extracellular immunoglobulin G (IgG) domains for ligand binding and a catalytic tyrosine kinase sequence in the intracellular domain. Each receptor activates several signal transduction pathways. The extracellular portion of p75 contains four cystein-rich repeats, and the intracellular part contains a death domain. Neurotrophin binding to the p75 receptor mediates survival, cell migration and myelination through several signalling pathways. Interactions between Trk and p75 receptors can lead to changes in the binding affinity for neurotrophins. BDNF, brain-derived neurotrophic factor; JNK, Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; NGF, nerve growth factor; NT, neurotrophin; PI3K, phosphatidylinositol 3-kinase; PLC-γ, phospholipase Cγ.
Chao (2003) Neurotrophins and their receptor: a convergence point for many signalling pathway. Nature Review 4, 299-309
The major site at which neurotrophins interact with these receptors is in the membrane-proximal immunoglobulin-like domain. The ability of Trk and p75 receptors to present different binding sites and affinities to particular neurotrophins determines both their responsiveness and specifity. The ratio of receptors is important in dictating numbers of surviving cells, and interactions between p75 and Trk receptors provide greater discrimination between different neurotrophins.25
The effects of neurotrophins on axon guidance can also be modulated by the intracellular location of the neurotrophin-receptor complex. During development, neurotrophins are produced and released from the target cells and become internalized into vescicles, which are then transported to the cell body. The biological effects of neurotrophins require that signals be conveyed over long distance, from the nerve terminal to the cell body36. Both Trk and p75 receptors undergo retrograde and
anterograde transport. Several proteins are associated with the Trk and p75 receptors during transport, and signalling persists after internalization.37
1.2 ROLE OF NGF IN THE DORSAL ROOT GANGLION (DRG)
In humans, there are 31 right and left paired “mixed” spinal nerves carrying autonomic, motor, and sensory information between the spinal cord and the periphery, 8 cervical spinal nerve pairs, 12 thoracic pairs, 5 lumbar pairs, 5 sacral pairs, and 1 coccygeal pair. These spinal nerves, formed from afferent sensory dorsal axons (the dorsal root) and motor ventral efferent axons (the ventral root), emerge from the intervertebral neural foramina between adjacent vertebral segments38,39,40. As the dorsal sensory root exits
the neural foramina, it forms the DRG, a collection of bipolar cell bodies of neurons surrounded by glial cells and the axons of the DRG sensory cells that form the primary
afferent sensory nerve. Because DRG neurons have two branches that act as a single axon, a distal process and a proximal process, connected by a cell body as an offshoot, they are called pseudounipolar neurons to differentiate them from bipolar cells, where the body is intersperced between two axons. (Figure 2)41
Figure 2 - 1) A pseuodunipolar sensory neuron. A pseudounipolar neuron has one axon that is divided into two separate branches, one from the periphery to the body and one from the body to the spinal cord. There are no dendrites. Unipolar cells are not to be confused with bipolar cells (2) where the body lies within the path of the axon. Unipolar cells have a T-stem axon that is away from the main axon.
Krames, S. E. (2014) The Role of the Dorsal Root Ganglion in the Development of Neuropathic Pain. Pain Medicine 15, 1669– 1685.
The DRG contains the greatest proportion of the body’s sensory neurons, cells that are primarily responsible for the transduction of sensory information from the periphery and transmitting the information to the central nervous system (CNS). The cell bodies, previously thought to be only metabolic storage “helpers” to processes that occur in the periphery that include nociception, are now known to actually participate in the signalling process by sensing certain molecules and manufacturing other molecules that modulate the process42. The cell bodies of DRG neurons do not interact with one
another. Because of its important role in the modulation of peripheral and central sensory processing that includes nociceptive pain, and the development of NP, and
pedicles38,39,40, the DRG is an excellent clinical target for pain control from both the
outside into the epidural space through the neural foramen and from the epidural space to the outside (Figure 3). The DRG is a known clinical target for the delivery of anti-inflammatory steroids43,44, for surgery (ganglionectomy)45, for radio-frequency
ablation46,47,48, for pulsed-radio frequency49, and for electrical stimulation
(neuromodulation)50,51,52.
Figure 3 - A cartoon of a section through the cervical intravertebral foramen showing the position of the DRG outside of the intervertebral neural foramen and its relationship to the IT space, the neuroforamina, the epidural space and dorsal and ventral roots. Krames, S. E. (2014) The Role of
the Dorsal Root Ganglion in the Development of Neuropathic Pain. Pain Medicine 15, 1669–1685
During ontogeny, primary sensory neurons are dependent on the presence of neurotrophins, in particular NGF, which plays a pivotal role in the regulation of axonal growth and guidance through target-derived synthesis, or by the determination of physiological cell death5,53. Experimental NGF administration in E14–E19 rat embryos
leads to an increased volume of small to medium-sized cells in dorsal root ganglia (DRG), as well as to sprouting of axons in layers of the spinal cord that primarily integrate noci- and proprioceptive as well as mechanoreceptive functions, whereas nerve fibers that travel to laminae 3/4 and to motoneurons do not respond to NGF54.
receptor, trkA56, results in the loss of 70-90% of DRG cells. Postnatally, dependence of
DRGs on NGF decreases as early as postnatal day 257; however, in vitro and in vivo
studies have shown that subpopulations of DRG neurons continue to express receptors for NGF, such as trkA and p75 neurotrophin receptors58,59,60,61. The trkA-positive DRG
cells in the adult represent ca. 40% of the entire DRG cell population and belong to the group of small to medium-sized cells that are phenotypically characterized by their expression of neuropeptides, in particular, calcitonin gene-related peptide (CGRP).62
Consequently, physiologic functioning of these neurons, including neurotransmitter regulation, depends on the presence of NGF. It has been shown that the two main transmitters of the peripheral nociceptive system, the neuropeptides CGRP and substance P, can be upregulated by NGF63,64,65,66,194, whereas in a small, but functionally
significant population of trkA-positive ganglia cells, the expression of vasoactive intestinal polypeptide (VIP), cholecystokinin (CCK), neuropeptide Y (NPY), and/or galanin is suppressed by the neurotrophin67. This mechanism is thought to be part of the
regulatory system that controls transmitter balance and may be of importance under pathological conditions, i.e., acute degeneration in the sensory system where initially a reduction of CGRP and substance P and an increase in the other peptides is found68.
The physiologic source of NGF for DRG cells is provided by local synthesis in Schwann cells or by expression in the peripheral innervation area. NGF reaches the soma by retrograde transport after intracellular uptake59. Recent evidence supports the
‘signaling endosome model’ in retrograde signaling. In this mechanism, target-derived neurotrophin binding to Trk receptors first induces receptor activated signaling in the local neuron terminal. This local signaling then causes the neurotrophin–receptor complexes to be internalized by endocytosis. Some of these endosomes re-configure
within the cell, so that activated Trk receptors are within the vesicle membranes and the neurotrophin is within the lumen of the vesicle. Such an orientation maintains the activated Trk tyrosine kinase domains in the cytoplasm, where they can interact with downstream-signaling effectors during transport through the axon, and ultimately, at the cell body. In the case of NGF binding to Trk A, the endocytotic retrograde transport system will permit both quick signaling at the nerve terminal, a sustained signaling through axonal transport, and a long-lasting signal that is still active by the time the endosome reaches the cell body.36Although the mechanism of neurotrophin retrograde
transport is only recently reported, there is ample evidence that it is altered in the diabetic state. NGF transport is depressed in the mesenteric nerves of STZ-induced diabetic rats; these nerves subserve the alimentary tract and may be prone to distal axonopathy induced by diabetic insult69. STZ-induced diabetic rats also show reductions
of 50% in NGF transport via the sciatic nerve, and NGF receptor saturation decreased by 45% compared to control rats. Thus, diabetic peripheral neuropathy also suppresses axonal transport in somatic sensory neurons70,71.
However, NGF receptors are not restricted to DRG neurons, but are also present in the projection area within the spinal cord (lamina 1/2), suggesting a modulatory role of NGF in the sensory pathways also at the level of the central nervous system58. In
addition, neurotrophins exert their influence not only along the central direction of the neuronal pathway of the sensory system, but also within the receptive field of the primary sensory neuron, i.e., receptors in the skin, Aδ-fibers for mechanonociception or C-fibers for thermonociception. A previous study has demonstrated that collateral sprouting of Aδ- and C-fibers in the partially denervated skin is supported by endogenous NGF and can be blocked by the application of NGF antibodies, whereas
regrowth of crushed nociceptive axons occured surprisingly independent from NGF72,
suggesting that the cells possess additional properties for the induction of regenerative mechanisms.62
1.3 NGF AND PAIN
Evidence that NGF has a key role in the generation and potentiation of pain is derived from experimental and clinical studies in a wide variety of acute and chronic pain states.73 NGF levels are elevated in several painful conditions in humans, including
arthritis74,75, cystitis76,77, prostatitis78 and chronic headaches79. Experimental animal
models of pain offer the advantage to study pathological mechanisms in more detail80.
For example, peripheral joints with nociceptors, the spinal cord and the CNS are all available for concomitant analyses of neuropeptides. Moreover, animal strains allow a more uniform examination as compared to genetically variant humans. Tissue analyses of experimental diseases and animal behavior do not always correlate with the targeted human disease. Nevertheless, animal studies provide valuable insights into immunological events and thus may give insight into human diseases.81
Similar to NGF-upregulation in human rheumatic diseases82, animal studies show
involvement of this molecule in experimental pain models. For example, NGF immunoreactivity was shown in dorsal spinal ganglia in experimental radiculopathy83.
A similar overexpression of neuropeptides including NGF is found in animal models with experimentally induced arthritis84,85,86 and OA in dogs87. These robust NGF-related
reactions following pain and inflammation suggest that anti-NGF regimens may result in a reduction of pain perception. Indeed, several animal studies have shown impressive anti-inflammatory and analgesic effects88,89,90. In particular, pain behavior after partial
ligation of the sciatic nerve was significantly reduced after intraperitoneal injection of anti-NGF antiserum91. In an experimental fracture pain model with C57BL/6J mice, an
anti-NGF antibody also showed significant pain reduction92. MNAC13 is an anti-TrkA
monoclonal antibody that exhibited similar potent analgesic effects in formalin-evoked pain licking responses in mice93. One experimental OA model94 indicates comparable
analgesic effects of an anti-NGF regimen using the TrkA domain 5 (TrkAd5) protein, a soluble receptor with high affinity to NGF95.
Induction of the expression of NGF is an early event in injured and inflamed tissues, and elevated levels of NGF are sustained in chronic inflammation. These changes in the synthesis of NGF appear to be caused, in part at least, by the action of pro-inflammatory cytokines, many of which induce the synthesis NGF in several cell types in vitro and in vivo96. NGF sensitizes nociceptive neurons directly to several pain-provoking stimuli by
causing rapid post-translational changes in the transient receptor potential vanilloid receptor 1 (TRPV1) cation channel and by modulating the expression of genes that influence nociceptor function. NGF also sensitizes nociceptors indirectly by activating mast cells. (Figure 5). Evidence indicates that trkA receptors are required for the nociceptive actions of NGF, but that p75NTR might also have a role.73 TRPV1 is a
cation channel that was identified originally as the capsaicin receptor. It is expressed on polymodal nociceptors and serves as a molecular detector for noxious heat and extracellular acidification, which occurs in tissue inflammation. When activated, TRPV1 enables the influx of monovalent and divalent cations, predominantly Ca2+,
Figure 4 - The main ways in which nerve growth factor (NGF) promotes pain and hyperalgesia following inflammation. The local production of inflammatory cytokines such as interleukin 1 (IL-1) and tumour necrosis factor (TNF) promotes the production of NGF by several cell types in the vicinity. NGF binds to trkA receptors on a major subset of nociceptive terminals. This triggers localized post-translational changes in TRPV1 that increase excitability. Retrograde trafficking of signalling endosomes to the cell body results in enhanced expression of proteins that further increases excitability and facilitates the activation of second-order neurons in the CNS. Abbreviations: ASIC3, acidsensing ion channel 3; BDNF, brain-derived neurotrophic factor; BK, bradykinin; HA, histamine; PG, prostaglandin; SP, substance P. (Hefti, F. F. et al (2006) Novel class of drugs based on antagonism of NGF. Trends in Pharmacological Sciences 2, 85-91)
In culture, NGF rapidly potentiates the activity of TRPV1 channels in DRG neurons treated with capsaicin98,99. Studies using pharmacological inhibitors in these neurons
indicate that the PI3K pathway is crucial for mediating sensitization to NGF, with both Ca2+–calmodulin-dependent kinase II and protein kinase C (PKC) acting downstream of
PI3K99,100. PKC-ε sensitizes TRPV1 by direct phosphorylation, which leads to increased
channel activity101,102,103 and translocation of the channel to the cell surface104.
Accordingly, NGF-induced hyperalgesia is inhibited by a PKC-ε-selective peptide inhibitor105. Retrograde NGF signalling from the peripheral terminals to the cell bodies
of nociceptive neurons106 enhances the expression of several proteins that further
sensitize these neurons and facilitate activation of second-order neurons in the CNS. These include: (i) substance P, which acts at central synapses to increase the activity of
second-order nociceptive neurons107; (ii) the Nav1.8 Na+ channel, which is expressed
exclusively by nociceptors and is implicated in NGF-induced hyperalgesia96,108,109; (iii)
the acid-sensing ion channel 3, which is activated by protons in ischaemic and inflammatory pain110; (iv) the TRPV1 channel111; and (v) BDNF112, which is
transported to central synapses where it increases nociceptive spinal-reflex excitability113. Retrograde signalling by NGF also increases the anterograde transport of
TRPV1 from the cell body to the peripheral terminals of nociceptors114. The three major
MAPK families, ERK, p38 MAPK and c-JUN N-terminal kinase, are induced by NGF in sensory neurons and are implicated in NGF-promoted hyperalgesia100,114,115. Because
activated MAPKs are present in the NGF–trkA signalling endosomes that are transported retrogradely to the cell bodies of sensory neurons106, it is possible that
activation of downstream transcription factors by these MAPKs contributes to the transcriptional changes in nociceptive neurons that are associated with peripheral and central sensitization. Exposure of isolated mast cells to NGF and lysophosphatidylserine (a molecule on the surface of activated platelets), but not to either factor alone, induces the release of 5-hydroxytryptamine (5-HT)116. This indicates that NGF sensitizes mast
cells under conditions of tissue injury and inflammation. In addition to 5-HT, activated mast cells release other pain mediators such as prostaglandins, bradykinin, histamine, ATP, HC and NGF itself, which stimulate nociceptor terminals and potentiate the pain response116. This positive-feedback loop is likely to contribute to the pain associated
1.4 ROLE OF NGF IN BONE METABOLISM
NGF appears to evoke response in certain non-classical neural tissues, e.g. embryonic cartilage rudiments117. Bone is constantly remodelled and its maintenance requires a
coordinated equilibrium between bone formation and bone resorption. Remodelling is influenced by a multitude of local and systemic factors acting either directly or indirectly on bone cells118. Rat osteoblastic-like cells (osteosarcoma cells) possess
receptors for thyroid hormone119, nerve growth factor (NGF)120 and norepinephrine
(NE)121, which suggests that these factors are capable of acting directly on osteoblastic
cells and could therefore play a role in bone remodelling. NGF, demonstrated by immunohistochemistry during craniofacial development in the mouse, appears in premuscular and precartilaginous mesenchyme as well as in the teeth.122 Cartilaginous expression of NGF supports the view that this molecule could play a role in the regulation of preskeletal differentiation.123 Moreover, Frenkel et. al showed that NGF is not only present in chick embryo cartilage, but also in bone.124
In bone morphogenesis, bone-associated neurons have been considered to regulate bone differentiation through synaptic interaction between neuronal cells and bone forming cells.125 Furthermore, Nakanishi et al126 suggest that osteoblast-derived neurotrophins may support not only survival and differentiation of neural cells, but also the proliferation of osteoblasts themselves in bone tissue in vivo, finally leading to bone formation. During bone proliferative phase and differentiation there is an increase in the level of NGF mRNA127. Interestingly, it has been observed that the presence of neurons increased thymidine incorporation into non-neuronal cells by up to 400 %128. These results suggest that NGF could induce osteoblastic cell stimulation and may reflect the
progressive interaction between bone cells and bone-associated neurons in the bone differentiation phase127.
After a bone fracture, neurotrophins and their receptors appear to be also involved in skeletal cell proliferation and differentiation. At 1 and 3 weeks after fracture in rat ribs, NGF was found to be widely localized in fracture callus periosteal osteoprogenitor cells, marrow stromal cells, osteoblasts, certain chondrocytes, young osteocytes and endothelial cells of new capillaries129.
1.5 OSTEOARTHRITIS
Osteoarthritis (OA) is an age-related chronic degenerative joint disorder, increasing in prevalence as the population ages130. It is the most common form of joint disease with
over one-half of all people older than 65 years of age demonstrating radiographic changes of OA in the knees.131 Clinical symptoms are primarily pain coupled with joint
stiffness and dysfunction, but the origin of this pain is unclear and although there is an evident inflammatory component, OA is not considered to be an inflammatory disorder.132 The slowly developing breakdown cartilage in the joints occurs
concomitantly to changes in the subchondral bone, synovium and muscle. As the cartilage is not innervated133, pain in OA is likely due to changes in the underlying bone
and inflammation in the synovium.
OA is a complex disease whose pathogenesis includes the contribution of biomechanical and metabolic factors which, altering the tissue homeostasis of articular cartilage and subchondral bone, determine the predominance of destructive over productive phenomena. The actors of this scenario are chondrocytes and osteoblasts which, in physiologic settings, regulate tissue differentiation and growth and which, during OA, increase the production of pro-catabolic cytokines (not counterbalanced by
adequate synthesis of inhibitors of pro-catabolic cytokines and growth factors), synthesize an inefficient ECM, and activate the enzymatic cascade leading to tissue destruction.134 However, chondrocytes produce chemokines, cytokines and proteases which can sensitize primary afferent fibre terminals in adjacent tissues135 and recent
evidence indicates that cartilage vascularization can result in sensory nerves growing along new blood vessels in osteoarthritic joints.136
Currently, there are no commercially available drugs definitively proven to alter the natural progression of this disease in the clinic.137 The supplements, glucosamine and chondroitin sulfate, however, may have the ability to provide chondroprotective effects and improve some of the signs and symptoms associated with OA138,139. In the absence of disease modifying drugs, the treatment of patients with OA is often directed at relieving pain and restoring function through the use of pharmacologic therapies140,141,142. Acetaminophen and nonsteroidal anti-inflammatory drugs (NSAIDs) are widely prescribed for the treatment of OA pain. The long-term use of these agents, however, may be accompanied by a number of undesirable effects including suppression of platelet aggregation and disruption of the gastrointestinal mucosa, the latter effect frequently leading to upper gastrointestinal erosions and ulceration143. Intraarticular injections of corticosteroids have been demonstrated to relieve inflammation and associated pain, but their effect is of short duration and is therefore employed infrequently140. For these reasons, it is apparent that the need exists for new therapeutic agents with the ability to attenuate OA-associated pain.
1.5.1 NEW THERAPIES FOR OA
In adults, injection with NGF results in pain, a finding that motivated the development of a NGF antagonist approach for clinical pain modulation (Figure 5). NGF-related therapies currently involve the use of proteins such as antibodies, peptibodies, and small protein domains, which either sequester NGF or prevent it binding to TrkA.144
Figure 5 - Transmission of pain by NGF. The neurotrophin β-nerve growth factor (NGF) is released upon injury and causes pain by activating its receptor (TrkA) on nociceptors and mast cells (which causes the release of inflammatory mediators such as histamine), leading to transmission of pain signals from the periphery to the spinal cord and brain via the dorsal ganglion. Inhibitors of NGF have shown promise in clinical trials of musculoskeletal pain.
Lane, E. N. and Corr, M. (2017) Osteoarthritis in 2016 - Anti-NGF treatments for pain — two steps forward, one step back? Nature Reviews, Reumathology.
In individuals with musculoskeletal pain, treatment with NGF inhibitors can produce impressive improvements in joint pain and physical function; however, mild neurologic adverse events and cases of accelerated arthropathy have been reported in clinical trials, leading to suspension or delay of investigations into these agents.144 Sequestration of
models of inflammatory and neuropathic pain, with resulting attenuation of pain-related behavior.144 The rat anti-NGF monoclonal antibody (mAb) αD11145 deserves a special
interest, as a therapeutic candidate, because it binds mouse NGF (mNGF) with picomolar affinity146 with no cross-reactivity towards closely related members of the
neurotrophin superfamily147 and antagonizes very effectively its biological function in a
variety of in vitro and in vivo systems148,149,150,151,152. This antibody has been
investigated in preclinical animal models of inflammatory and chronic pain153,154.
In addition to the sequestration of NGF, blocking the effects of NGF trough TrkA can also be achieved by blocking the effects of NGF/TrkA interaction on the receptor itself.144 A monoclonal antibody to TrkA, MNAC13 has proved effective in attenuating
pain responses in models of both inflammatory and neuropathic pain. This antibody blocks the interaction between NGF and TrkA, and is specific for TrkA: it does not bind the other Trk receptors.144
Side effects of NGF-antagonists are different from those observed with conventional NSAIDs and include peripheral edema, joint swelling, myalgia, arthralgia, pain in extremity, back pain, carpal tunnel syndrome, hypoesthesia, hyperesthesia, and paresthesia. Urinary tract infection, nausea, constipation, nasopharyngitis, osteoarthritis, hypertension, and headache have all been reported155,144. Less often, peripheral
neuropathy and allodynia have been observed. Severe adverse effects with low incidence have included atrial fibrillation, coronary artery disease, chest pain, bacterial cellulitis, pneumonia, fall, breast cancer, bone fractures, or pulmonary embolism. NGF regulates axonal regeneration after neuronal injury in the adult. This is true for peripheral polyneuropathy156 and possibly also for recovery from ischemic injury of the
disease159,160, or multiple sclerosis161. Neutralizing antibodies may thus reduce NGF
concentrations at sites of healing in the periphery and in the central nervous system in cases of an incomplete blood–brain barrier. In the peripheral nervous system of animals with experimentally induced arthritis, however, anti-NGF treatment inhibited sprouting of sensory and sympathetic nerve fibers without any effect on inflammatory infiltrates with CD31+ aberrant neovascularization or CD68+ macrophages162.
Bone remodeling seems to be another important consideration in patients treated with NGF-antagonists. Anti-NGF treatment reduced bone resorption in ligature-induced periodontitis with a concomitant reduction of gingival IL-1β expression163. In 2010,
clinical trials were placed on partial clinical hold by the Food and Drug Administration (FDA) after several suspected cases of osteonecrosis occurred both in osteoarthritis patients treated with NGF antagonists alone or with NSAIDs and with NSAIDs alone. Most of these cases were diagnosed as either RPOA, normal progression of OA, subchondral bone fractures, and others. RPOA may be induced by a variety of conditions and is characterized by subchondral bone loss and joint destruction within six to 18 months164. The incidence of RPOA in anti-NGF trials increased with
concomitant use of NSAIDs and was similar to those for patient cohorts in other studies. NGF antibodies downregulate proinflammatory, and secondary neurotransmitters such as SP or CGRP, and anti-NGF regimens could thus potentially induce systemic immunosuppression. To date, however, clinical trials have not resulted in substantially increased incidence of infection, suggesting that the immune system may bypass NGF-downregulated local mediators. Conversely, locally or systemically elevated levels of NGF have been observed in some allergic conditions, for example asthma, atopic
dermatitis, rhinitis, and contact eczema165 and these disorders may improve if the local
NGF is removed.155
1.5.1.1 αD11
αD11 is an anti-NGF rat antibody. It binds to mouse NGF with picomolar affinity and human NGF as well166. The epitope recognized by mAb αD11 was identified in Loops I
and II of mNGF. While the sequences of rat and human NGF in this region are identical, for the mouse and human NGF they differ in one position. A structural alignment of Loop I and Loop II of the mouse and human NGF crystallographic structures167,168 respectively, shows a good superposition. Thus, it can be reliably
predictable that mAb αD11 binds to hNGF equally well as to mNGF. Indeed, an ELISA assay, with solidphase coated mNGF and hNGF and serial dilutions of mAb αD11, had confirmed that mAb αD11 recognizes hNGF and mNGF with a comparable affinity166.
This antibody was humanized and it is currently testing in patients. Its analgesic effect was assessed in vivo with the formalin-induced pain test and the Chronic Constriction Injury (CCI) of the sciatic nerve one.166
In the first one, mice were injected with formalin that leads the animals to lick the site of injection. Thus, the effects of an administration of αD11 were assessed and αD11-treated mice showed a decrease in this behaviour respect to saline-αD11-treated ones.166
Figure 6 - In vivo analgesic activity of parental mAb αD11 in inflammatory pain model. Analgesic effects of αD11 antibody administration on the late phase (15–40 min) in the course of the formalin test. Treatment consisted in saline (negative control) or antibody injection (single doses: 12.5 mg of mock mouse mAb or two different molecular formats of αD11, i.e. mAb or Fab) performed (in the same paw as for formalin) 45 min before formalin injection and testing. Statistical analysis was performed on each phase (ANOVA and Fisher’s Test for comparison of each couple of groups). Each experimental group included at least 8 animals. [166]
The analgesic potency of mAb αD11 was further evaluated in a mouse model of neuropathic pain, the Chronic Constriction Injury (CCI) of the sciatic nerve following two treatment protocols, a shorter and a longer lasting one. In both protocols, exhibited a very significant analgesic effect, as compared to mouse IgG mock. In a first set of experiments (short protocol) (Figure 7B), four I.P. injections of mAb αD11 (from day 3 to day 6 after ligation of the nerve) were able to significantly reduce mechanical allodynia, starting from day 4 after surgery. On this basis, a second set of experiments with a longer observation period (long lasting protocol, observation up to 31 days following sciatic nerve ligature), was performed. The observation of animals undergoing long lasting protocol revealed a quite unexpected temporal profile for the strong analgesic activity induced by mAb αD11 (Figure 7C). Two phases can be
recognized in the analgesic action: the first one identifies a pharmacological effect of mAb αD11 (an effect which declined in parallel with the drop of the antibody concentration in circulating blood, reaching a minimum analgesic effect around day 17, i.e. one week after the end of the treatment). After the gradual decline of the anti-allodynic effect, in the second phase (from day 21 to day 31) mAb αD11 again reduced neuropathic pain, displaying a longterm analgesic effect of the anti-NGF antibody (Figure 7C). This long lasting analgesic effect is likely to involve persistent changes in gene expression in sensory neurons, demonstrating that αD11 antibody is not just as a potent analgesic, but also a long-term disease-modifying drug.166
Figure 7 - In vivo analgesic activity of parental mAb αD11 in neuropathic pain models. (a) Analgesic effects of αD11 antibody administration on the late phase (15–40 min) in the course of the formalin test. Treatment consisted in saline (negative control) or antibody injection (single doses: 12.5 mg of mock mouse mAb or two different molecular formats of αD11, i.e. mAb or Fab) performed (in the same paw as for formalin) 45 min before formalin injection and testing. Statistical analysis was performed on each phase (ANOVA and Fisher’s Test for comparison of each couple of groups). Each experimental group included at least 8 animals. (b) Analgesic effects of αD11 antibody in the short lasting protocol of neuropathic pain model: mAb αD11 significantly increased the value of ipsilateral/contralateral index (ratio between the threshold forces measured for the two hind paws, the one ipsilateral to surgery and the contralateral one. Mean value 6 s.e.), starting from day 4 to day 14, one week after the last antibody injection. Control mice were injected with either mock mouse IgG, (1.4 mg/Kg) or saline solution (sal). ANOVA test for repeated measures resulted in statistical significance for treatment (p,0.0001), time (p,0.0001) and the interaction between the two factors (treatment time) (p,0.0001). (c) Analgesic efficacy of mAb αD11 (one dose: 2 mg/kg) in the long lasting protocol of neuropathic pain model. MAb αD11 increased the ipsilateral/contralateral index, starting either from day 5. The analgesic effect, which disappeared around days 17–19, increases again to reach a plateau between day 27 and day 31, identifying a late phase in the action of mAb αD11 (long-term effect). ANOVA test for repeated measures resulted in statistical significance for treatment (p,0.005), time (p,0.005) and the interaction between two factors (treatment6time) (p,0.005). [166]
1.5.1.2 MNAC13
MNAC13 (Figure 8) is a mouse antibody that binds to rat and human trkA as well. It was isolated thanks to mouse immunization.169
First, to produce antibodies able to interfere with the neurotrophin binding activity of the TrkA receptor, a congenic immunization protocol was exploited. BALB/C-3T3 cells expressing the human TrkA receptor (TrkA-BALB/C 3T3), produced by transfection of the human trk proto-oncogene, were used for immunization of BALB/C mice. Hybridoma supernatants were screened by a functional assay, namely their ability to inhibit the binding of 125I-NGF to BALB/C 3T3-TrkA cells. Of 1266 wells in which hybridoma growth was occurring, only four supernatants showed NGF neutralizing activity. The corresponding cells were subcloned, giving rise to clones MNAC13, MNAC30, MNAC191, and MNAC232. The ability of the antibodies produced by these clones to inhibit the binding of NGF to TrkA-BALB/C 3T3 cells is demonstrated in Figure 10. These anti-TrkA antibodies inhibit the binding of NGF as efficiently as the neutralizing anti-NGF antibody αD11 (Figure 9).
Figure 8 - TheMNAC13 antibody (here represented as monovalent antigen binding fragment) binds to TrkA extracellular domain (ECD), preventing TrkA from being activated by its natural ligand NGF. 93.
Figure 9 - Inhibition of binding of 125I-NGF to TrkA-BALB/C 3T3 cells. Hybridoma supernatants were preincubated with TrkA-BALB/C 3T3 cells before the addition of 125I-NGF. The histogram reports the inhibition of specific NGF binding to TrkA-BALB/C 3T3 cells by different antibodies. Specific binding was evaluated by subtracting from the total binding that obtained in the presence of an excess of unlabeled NGF. [169]
To confirms that mAb MNAC13 binds specifically to the TrkA, an ELISA assay was performed (Figure )
Figure 10 - mAb MNAC13 recognizes the extracellular domain of TrkA receptor and does not inhibit NGF binding to soluble TrkA receptors. [169]
To demonstrate the efficacy of MNAC13 in blocking trkA signal, hybridoma cells secreting the MNAC13 antibody were implanted in the lateral ventricle of neonatal rats 2 days after birth, and the cholinergic phenotype of the neurons was studied 1 week later by immunohistochemistry with antibodies against ChAT. The forebrain is rich of cholinergic neurons that are target for NGF action in the CNS.170
Figure 11 - Implant of mAb MNAC13-secreting cells in the rat brain (b) significantly reduces the number of cholinergic basal forebrain neurons respect to control (a). [183]
The results had shown that the number of ChAT-positive cells was dramatically reduced in the brains implanted with the anti-TrkA antibody (Fig. 11B), with respect to controls (injected with a nonrelevant myeloma) (Fig.11 A).
The analgesic effect of MNAC13 was assessed by the same tests used for αD1193. The
licking behaviour decreased in MNAC13-treated mice respect to control in the formalin-induced pain.93 (Figure 12)
Figure 12 - Analgesic effects of MNAC13 on inflammatory pain in CD1 mice on the early (0–15 min) and late (15–40 min) phase of formalin test [93]
The efficacy of MNAC13 was assessed also in the neuropathic pain model where, also in this case, leads to an improving in the pain perception both in the smaller and in the longer lasting protocol.93 (Figure 13)
Figure 13 -
Antiallodynic effects easured with a dynamic plantar aesthesiometer after four i.p. injections of 50 ug per mouse MNAC13 (from day 3 to day 6 after ligation of the sciatic nerve) in CCI mice tested for 14 days (A) and after eight i.p. injections of 30 or 70 ug per mouse MNAC13 (from day 3 to day 10 after ligation of the sciatic nerve) in CCI mice tested from day 3 up to day 31 (B).93
1.6 PAINFUL DIABETIC NEUROPATHY
Painful diabetic neuropathy (PDN) is a common complication of both type 1 and type 2 diabetes. PDN is an early manifestation of diabetic neuropathy and frequently precedes the diagnosis of diabetes171,172. Several recent studies have suggested that nearly
one-third of the patients with impaired glucose tolerance (pre-diabetes) seek medical attention for a pain syndrome identical to PDN173. While PDN is a persistent symptom
in epidemiological studies of patients with type 2 diabetes, it is less common in type 1 diabetes174,175,176,177; while estimates vary, approximately half of all patients with
diabetic neuropathy and type 2 diabetes experience PDN, usually at the onset of their disease. Patients with PDN experience allodynia and hyperalgesia; allodynia occurs when normally non-painful stimuli become painful, whereas hyperalgesia is increased sensitivity to normally painful stimuli. PDN is a major factor in decreased quality of life for patients with diabetes178,179. Over a period of time that could last several years, PDN
subsides and the disabling pain is replaced by a complete loss of sensation, leading to the numb, insensate diabetic foot172,180. In order to understand the pathogenesis of
diabetic neuropathy, one must first consider the neurons that are at risk in this disorder and then address the qualities about these cells that make them vulnerable. In diabetic peripheral neuropathies, the symptoms always first appear in a stocking distribution. The vulnerability of these neurons is due to the length of their axons.181 Neurons that
form the sensory relays of the spinal cord to the feet have the longest axons amongst any other neurons in the body. For illustration, the neuronal axon from the spinal cord to the foot in a tall individual may be over 1m long. These long axon-bearing sensory neurons are the most vulnerable targets in diabetic peripheral neuropathy.181 The most
either positive (pain, excessive sensitivity to temperature) or negative (numbness, loss of sensory perception).182 Sensory peripheral neuropathy most often affects the feet and
legs, although with disease progression, the hands and arms may also be affected. Loss of sensation in the feet is the most common effect of diabetic neuropathy and promotes significant risk to the limb. Due to decreased tactile and pain perception, injuries to the feet are often undetected by the patient and thus may go unchecked and untreated. Successive injuries manifest as non-healing foot ulcerations, and progressive damage to the feet can ultimately result in severe infection, gangrene, and amputation.183
The past 20 years of research have investigated the pathogenesis of diabetic peripheral neuropathy, seeking to elucidate points for therapeutic intervention. Although the pathogenetic mechanism is not fully understood, the disorder is pathologically marked by degeneration of Schwann cells and myelinated neuronal fibers as well as loss of a population of the neurons located in the dorsal root ganglia (DRG).184,185,186,187,188.
These trkA-expressing, NGF-responsive peptidergic DRG neurons are the key generators of neuropathic pain189,181 and are affected early in the course of diabetes189.
Two of the most extensively studied neuropeptides are SP and CGRP. SP is a tachykinin neuropeptide that mediates nociception and is used as a marker for pain in animal models190,191. Under normal conditions, SP is expressed only in
small-to-medium-sized trkA-positive DRG neurons190. During painful conditions, SP is
upregulated in these DRG neurons and released to the Lamina I and the outer layer of Lamina II of the spinal cord dorsal horn to activate secondary sensory neurons190,192.
CGRP is also upregulated under painful conditions in similar population of trkA-positive sensory neurons193. NGF is a major factor in enhancing the expression of SP
and CGRP194. Exogenous exposure to high levels of NGF, both in vivo and in vitro,
increases the intracellular content and release of SP and CGRP195,196,197,198. NGF may be
involved in mediating PDN via upregulation of SP and CGRP.199 Development of
neuropathic pain following diabetes may be accompanied by alterations in the level or distribution of these neuropeptides200. Understanding the role that NGF plays in
nociceptive peptide expression would set the stage for uncovering the mechanisms underlying the induction and modulation of neuropathic pain in type 2 diabetes199.
Alterations in the synthesis and expression levels of growth factors in the peripheral nervous system may account for the vulnerability of neurons and Schwann cells to the diabetic state. Most research has focused on the changes in NGF synthesis and expression in in vitro and in vivo models of diabetic peripheral neuropathy. These models include injury-induced nerve damage such as sciatic nerve transection, axotomy or crush, the streptozotocin (STZ)-induced diabetic rat, and the spontaneously diabetic BB rat. Following sciatic nerve crush, NGF expression is upregulated in DRG. TrkA is also expressed by DRG during this time, suggesting that DRG mount a self-directed regeneration attempt201. After axotomy of adult rat sciatic nerve, NGF, TrkA, and p75
NGF receptors are 50% downregulated in DRG at 4–14 days post-injury and do not return to control levels until 30 days post-injury202. However, NGF, TrkA, and p75 are
increased in Schwann cells distal to the sciatic nerve injury site, reaching maximal levels 5–7 days following the injury. In sciatic nerve injury models, NGF expression in Schwann cells is inversely related to axonal contact. For example, NGF expression increases in Schwann cells when proximal axons are degenerating, and expression is suppressed once regenerating axons grow in contact with the Schwann cells203. TrkA
model204. These results suggest that the NGF system may be upregulated in Schwann
cells in an attempt to promote their own survival and to provide NGF to nearby damaged neurons to promote their survival. Biopsies of human sural nerve recapitulate these results. NGF receptors are present in Schwann cells in sural nerves that contain degenerating axons, but these receptors are not detected in Schwann cells of nerves that have remyelinated205. Expression of the NGF system is also altered in rat models of
diabetes. In STZ-diabetic rats, NGF levels are decreased in sympathetically innervated target organs, the superior cervical ganglion, and sciatic nerve206. However, NGF
expression levels returned to normal following allogeneic pancreatic islet transplantation that provides a physiological glucose homeostasis without immunosuppression. The diabetic condition itself causes NGF reduction and return to euglycemia can restore normal nerve function207. Decreased NGF production also
occurs in genetically diabetic mice (C57Bl/KsJ db/db)208. Cheng et al suggest that NGF
levels are elevated during a period of mechanical allodynia at the early stage of diabetes in db/db mice. In this study they detected a transient increase in NGF immunoreactivity in the DRG neurons and hind paw skin in db/db mice. This phenomenon coincides with the development of mechanical allodynia, suggesting that NGF could play an important role in mediating pain in type 2 diabetes.199
There are little data about NGF expression in human diabetic patients, although these patients do exhibit increased NGF and TrkA mRNA in their lateral calf skin209,210. This
abnormal increase in skin may be the byproduct of a compensatory NGF production mechanism mounted to protect vulnerable NGF-responsive neurons. Furthermore, the p75 NGF receptor is upregulated in the sciatic nerves of patients with type 1 or type 2 diabetes mellitus211. However, others report depletion of skin NGF in patients with
diabetic neuropathy, which correlates with decreased skin axon reflex vasodilation responses mediated by small sensory fibers.212 Maybe that skin-derived NGF, normally
produced by keratinocytes, is important for maintaining nociception and thus loss of these cells and that NGF may lead to or exacerbate peripheral neuropathy.181 This
controversy regarding the changes in expression of the NGF system in diabetes in humans is intriguing, because different responses may underlie the variable susceptibility to and symptoms of neuropathy between patients.
1.6.1 TREATMENT OF PDN WITH NGF
Major components of the NGF signaling pathway have been found deregulated in experimental diabetes213,214,215, as well as the production of neuromodulators that is
known to be under NGF control216,217. On the other hand, NGF supply in animal models
of diabetic neuropathies reverses neuropathic signs, by protecting the affected PNS neurons and normalizing their activity218,219. The production of recombinant human
NGF (rhNGF) has been first developed and tested in phase I clinical trial, where moderate side effects, such as myalgias and injection site hyperalgesia, were evidenced in healthy subjects220,221. A phase II clinical trial on 250 patients affected by diabetic
polyneuropathy was then performed222,223. The study revealed a significant
improvement of neuropathic symptoms in the NGF-treated patients, but also evidenced the occurrence of side effects, such as injection site hyperalgesia, myalgias and arthralgias, that limited the blinding of the study222,223.
On these bases, an NGF “painless” mutant has been developed224 inspired by the HSAN
V mutation in the NGFB gene (Figure 14). HSAN V is a rare human genetic disease of congenital snsensitivity to pain, due to an aminoacid R to W substitution at position 100
of mature NGF protein225. hNGF R100E mutant maintains, in a variety of cellular
assays, identical neurotrophic and neuroprotective properties as the hNGF wild-type, while displaying a significant reduced pain-inducing activity in vivo224,226. This hNGF
mutants has therapeutic potential, aimed at the design of new clinical protocols for the treatment of different kind of diseases, such as Alzheimer's disease, diabetic neuropathies, ophthalmic diseases and dermatological ulcers, where the neurotrophic effects of NGF could be exploited, by avoiding the nociceptive side effects induced by the neurotrophin.224
AIMS OF THE RESEARCH THESIS
Experimental and clinical studies provide the evidence that NGF contributes to the generation and maintenance of a wide range of pain states. Thus, drug discovery efforts are focusing on the development of anti-NGF agents: several humanized anti-NGF monoclonal antibodies have entered clinical trials as potential analgesics. However, there are concerns about the development of side effects. A major side effect is the appearance of severe joint destructions that prompted the FDA to place such clinical studies on hold155.
In this thesis, with the aim of finding an alternative to anti-NGF antibodies, I compared the effects of NGF sequestration to the blockade of its receptor, TrkA, in a mouse model of osteoarthritis (OA).
I tested different ways of administration of the NGF antibody αD11 or of the anti-TrkA antibody MNAC13:
A multiple dose after induction of OA,
A single dose administered 3 days after OA induction,
A preventive administration constituted by a single dose of antibodies 24 hours before OA induction.
NGF has been shown also to be a potential drug to treat diabetic neuropathic pain. Degeneration of Schwann cells leads to a decrease of NGF levels and to a reduced innervation of patient feet and insensitivity to noxious stimuli. However, the administration of this protein induces intramuscular pain such as that the dosage used in clinical trials had to be reduced at such a point that the therapy was not efficacious223.
On this basis, I studied whether the administration of a NGF mutant (painless NGF, pNGF) characterized by the same neurotrophic activity of wild type NGF and reduced nociceptive activity could rescue the insensitivity to pain in a mouse model of diabetes.
MATERIAL AND METHODS
3.1 OSTEOARTHRITIS 3.1.1 ANIMALS
All experiments were carried out using male adult C57Bl/6 mice aged 8 weeks old (20– 25 g, Charles River, Calco, Italy). Food and water were available ad libitum and mice were housed under standard conditions with a 12-h light/dark cycle for 2 week prior to behavioural experiments to acclimatize. Experimental study groups were randomized and experiments were performed by an observer unaware of treatments.
3.1.2 INDUCTION OF OSTEOARTHRITIS
Mice were anaesthetized by isoflurane/O2 inhalation and received a single intra-articular injection into the right knee. Each mouse received vehicle or 1 mg monosodium iodoacetate (MIA) (Sigma, Gillingham, UK) in a total volume of 10 µL of physiological sterile saline. The right leg was flexed at a 90° angle at the knee and the solution was injected through the infrapatellar ligament into the right knee joint capsule using a 30-G needle and a Hamilton syringe (Bonaduz, GR, Switzerland).
3.1.3 ADMINISTRATION OF ANTIBODIES
- Anti-trkA antibody MNAC13 was administrated intraperitoneally in three different ways:
- Single dose before injection of MIA, 100μg. - Single dose after injection of MIA, 100μg.
- Anti-NGF antibody αD11 was administrated intraperitoneally in two different ways:
- Single dose before injection of MIA, 100μg. - Single dose after injection of MIA, 100μg.
3.1.4 INCAPACITANCE TESTER
Changes in hind paw weight distribution between the right (osteoarthritic) and left (contralateral control) limbs were utilized as an index of joint discomfort in the osteoarthritic knee. An incapacitance tester (Linton Instrumentation, Norfolk, UK) was employed for determination of hind paw weight distribution. Mice were placed in a Perspex chamber designed so that their two hindpaws were resting on separate transducer pads. (Figure 15) Once the mice were settled in the correct position, a reading of their weight distribution was taken over 3 s. Results are calculated as weight borne on the ipsilateral hindlimb as a percentage of total weight borne by the mouse:
Ipsilateral weight borne
Ipsilateral+controlateral weight borne x 100
A value of 50% represents an equal weight distribution across ipsilateral and contralateral hindlimbs and a value of less than 50% is indicative of a reduction in weight borne on the ipsilateral hindlimb. Two baseline trials were recorded prior to injection of MIA/vehicle. Mice were subsequently assessed on days 3, 7, 10, 14,17, 21 and 28 after injection. Each session consisted of three separate trials separated by at least 5 min.
Figure 15 - Incapacitance tester: mice are placed in an angled plexiglass chamber and are when the mouse is in the proper position (both hind paws on their respective force plates, both front paws on the plexiglass ramp, and facing forward) three, 3-s readings are recorded.
3.1.5 KNEE HISTOLOGY
At the end of the behavioural studies, 28 days after injection of MIA, animals were deeply anaesthetized, knees were dissected out and surrounding muscle was trimmed. Tissue sections were stained with hematoxylin and eosin or safranin by Levicept, Kent, England.
3.1.5.1 HEMATOXYLIN AND EOSIN
Hematoxylin has a deep blue-purple color and stains nucleic acids by a complex, incompletely understood reaction. Eosin is pink and stains proteins nonspecifically. In a typical tissue, nuclei are stained blue, whereas the cytoplasm and extracellular matrix have varying degrees of pink staining.
MATERIALS
Reagents
- Cells or tissue of interest on microscope slide (Step 1.i); - Eosin Y (1% aqueous solution; EM Diagnostic Systems); - Ethanol (95%, 100%);
- Methanol or Flex alcohols (Richard-Allan Scientific) can be used instead of ethanol (see Step 5);
- Hematoxylin, Mayer’s (Sigma);
- Mayer’s hematoxylin is the easiest to use and is compatible with most colorimetric substrates;
- Mounting medium (Canada Balsam, Sigma C1795);
- Use glycerol or other aqueous mounting media if alcohols cannot be used (see Step 7);
- Xylene.
METHOD
1. Hydrate the cells or tissue:
i. Use a microscope slide bearing cryosections or rehydrated tissue sections fixed in either alcoholor an aldehyde-based fixative ii. Immerse the slide for 30 sec with agitation by hand in H2O
A rinse in H2O is important; hematoxylin precipitates with salts and buffers. The staining can be performed after immunohistochemical or hybridization reactions with nonfluorescent detection systems. 2. Dip the slide into a Coplin jar containing Mayer’s hematoxylin and agitate for
