Chemotherapy induced peripheral neuropathy (CIPN) is a dose-limiting side effect of several antineoplastic agents commonly used in clinics. Up to now its management with common analgesic drugs is often unsatisfactory, therefore novel therapeutic approaches are needed. The molecular mechanisms underlying CIPN are still largely unknown, even if involvement of mitochondrial toxicity and oxidative stress, ion channels alterations and neuroinflammatory processes have been suggested (Boyette-Davis et al., 2015; Montague and Malcangio 2017). In particular, some cytokines and chemokines are involved in the initiation and persistence of pain (Sacerdote et al., 2013). Among chemokines, the Prokineticin family has an important role in immunomodulation, inflammation, nociception, inflammatory pain and in different experimental neuropathic conditions such as nerve lesion (Lattanzi et al., 2015; Guida et al., 2015) and diabetic neuropathy (Castelli et al., 2016). Both in humans and rodents, Prokineticin-1 (PK1) and Prokineticin-2 (PK2) (Negri et al., 2007) are ligands of two metabotropic receptors known as PK-R1 and PK-R2 (Masuda et al., 2002). Prokineticins and their receptors are expressed in the brain, spinal cord, dorsal root ganglia, granulocytes, macrophages and lymphocytes. It has been demonstrated that PK2 acts on macrophages and induces a pro-inflammatory profile (Martucci et al., 2006), which can lead to reduced nociceptive thresholds to thermal and mechanical stimuli (Negri et al., 2006; Giannini et al., 2009). Furthermore, in some nervous regions associated to pain, a co-localization of PK-Rs and TRPV receptors has been shown, and many neurons that respond to PK2 express and release typical pain mediators such as CGRP and substance P (Vellani et al., 2006). On the basis of these considerations, our study investigates the role of prokineticin system in a murine model of CIPN induced by the administration of two different chemotherapeutic agents commonly used in oncology and related to CIPN development: Vincristine (VCR), the most neurotoxic drug among Vinca Alkaloids, and bortezomib (BTZ), the first approved 26s proteasome inhibitor. These drugs, despite having different mechanisms of action on tumor cells, induce neurotoxicity and consequent neuropathy with similar mechanisms (Flatters et al., 2019; Boyette-Davis et al., 2015). C57BL/6J, 9 weeks old, male mice were used in all the experiments. Based on the most common protocols used in literature, CIPN was induced in mice through the intraperitoneal (i.p.) administration of bortezomib 0.4mg/kg, 3 times a week for 4 consecutive weeks (Boehmerle et al., 2014). Vincristine was administered at the dose of 0.1mg/kg, i.p. injected daily for 14 consecutive days (Kiguchi et al., 2008). Control mice were injected with the vehicle. Allodynia is one of the most frequent symptoms of CIPN, therefore, mechanical and thermal allodynia were monitored using Von Frey and Acetone Drop Test, respectively. Furthermore, thermal hyperalgesia was evaluated by Plantar test. Both bortezomib and vincristine induced in mice a painful neuropathy characterized by the presence of mechanical and thermal allodynia as well as thermal hyperalgesia. To assess the potential therapeutic effect of PC1, a non-peptidic antagonist of PK-Rs, BTZ- and VCR- mice in a clear hypersensitivity condition were subjected to chronic treatment with PC1 (150μg/kg, s.c. injected, daily administered for 14 or 7 consecutive days in the BTZ and VCR protocol respectively). Independently from the chemotherapeutic agent used to induce CIPN, PC1 was effective in ameliorating the painful symptoms. To assess the role of PK system in our model, for each experimental protocol (BTZ and VCR) we performed biochemical analysis at two different time points: before starting PC1 treatment (BTZ day 14 and VCR day 7), and at the end of chemotherapeutic schedule (BTZ day 28 and VCR day 14). Fourteen days after the first BTZ administration (BTZ day 14), no alterations were detected in PK system expression in the main stations involved in pain transmission (sciatic nerve, DRG, spinal cord). On the other hand, VCR-mice at day 7 (VCR day 7) already showed an upregulation of both PK-Rs at the level of the peripheral (DRG) and central (spinal cord) nervous system. Recruitment and infiltration of macrophages in DRG is known to have a key role in CIPN (Lees et al., 2017) and since PK2 is able to induce the macrophage to migrate and to acquire a pro-inflammatory phenotype, we evaluated expression of the macrophages/microglia markers (CD68 and CD11b) and of TLR4, whose activation is linked to pro-inflammatory cytokines modulation. At BTZ day 14 as well as VCR day 7, painful symptoms were related to an increase of CD68 and TLR4 expression. Immunofluorescence analysis confirmed the presence of activated macrophages in DRG and sciatic nerve of BTZ-mice. At the same time points, both BTZ- and VCR- mice peripheral nervous tissues showed a pro-inflammatory profile with high levels of TNF-α, IL-1β and IL-6 and low levels of IL-10. At BTZ day 14 and VCR day 7, the central nervous system was not directly affected by chemotherapy. At the end of the chemotherapeutic schedule (BTZ day 28 and VCR day 14), a stronger neuroinflammatory profile was detected in sciatic nerves and DRG of mice undergoing chemotherapy. The increased cytokine production could be sustained by infiltrating macrophages since we detected in both nerve and DRG of BTZ- and VCR- treated mice also an upregulation of CD68, CD11b and TLR4 and an increase of CD68 positive cells in BTZ-mice. These cells also expressed PK2, suggesting that infiltrating macrophages could be a source of PK2 in peripheral nervous tissues. At the same time point (BTZ day 28 and VCR day 14), also spinal cord was affected by chemotherapy-related neurotoxicity. In fact, an upregulation of all PK system components, high levels of CD68 and TLR4 and a clear pro-inflammatory cytokine profile were evident. Moreover, a colocalization between PK2 and the astrocytes marker GFAP in the spinal cord of BTZ-mice was observed, suggesting that astrocytes could be the main producers of the PK2 in this nervous station. An upregulation of both GFAP and CD68 was also present in the central nervous system of VCR treated mice. PC1 was able to counteract immune/neuroinflammatory activation and amplification, which is important to enhance hypersensitivity. In fact, the antagonist prevented or reduced the expression of CD68, CD11b and of TLR4 and restored a correct cytokine balance both in the peripheral and central nervous system. To more closely resemble the clinical situation, where patients are often subjected to several cycle of chemotherapy (Starobova & Vetter, 2017), mice previously treated with the chemotherapeutic agents, were subjected to a second BTZ- or VCR cycle. Following the interruption of chemotherapy, mice progressively recovered from neuropathy. In both protocols the second chemotherapy cycle induced again hypersensitivity, but the allodynic effect induced by the chemotherapeutic drugs was less evident in mice previously treated with PC1 if compared to that observed in BTZ/VCR-only re-treated mice, indicating a possible protective role of PC1. In the second part of our study, considering the susceptibility of DRG to chemotherapy (Montague and Malcangio, 2017), we began to study the involvement of neurons in CIPN using an in vitro model represented by DRG primary sensory neurons. In particular, we focused on neurite outgrowth, a morphological phenotype of neuronal cells that correlates with their function and health (da Silva et al., 2002). Neurons collected from wildtype mice were cultured and different concentrations of bortezomib and vincristine were tested (Alè et al., 2015). Both agents induced a dose-related reduction of neurite length compared to vehicle-treated cells. Different concentrations of PC1, based on the literature, (Severini et al., 2015) were co-applied to BTZ and VCR. PC1 dose-dependently prevented chemotherapy induced toxicity and the most effective dose of the antagonist was 250nM. We started to analyse biochemical parameters of neurons exposed to VCR alone or in association to PC1. VCR-cells showed an upregulation of PK2 and PK-R1, TLR4, IL-1β, IL-6, IL-10, and ATF3, which was not detectable in VCR-cells exposed also to PC1. VCR did not induce alterations in PK-R2, GFAP and TNF-α mRNA levels. PC1 250nM alone was tested in the cell cultures: no morphological/biochemical alteration was detected. Taken together our results show that PK system is involved in the development and maintenance of CIPN and, considering the positive effects of PC1 in contrasting both painful symptomatology and neuroinflammation, PK-Rs antagonism could represent a new therapeutic approach to handle this type of neuropathy.
La neuropatia periferica indotta da chemioterapici (CIPN) rappresenta uno degli effetti collaterali dose-limitante di numerosi agenti antineoplastici comunemente utilizzati in clinica. Il trattamento della CIPN, con i comuni farmaci analgesici è spesso insoddisfacente, pertanto è necessario lo sviluppo di nuovi approcci terapeutici. La patofisiologia della CIPN è molto complessa e non è stata ancora completamente chiarita; è caratterizzata da meccanismi multipatogenici tra cui: danno mitocondriale, stress ossidativo, alterazioni di canali ionici e neuroinfiammazione (Boyette-Davis et al., 2015; Montague e Malcangio 2017). In particolare, è stato dimostrato che le citochine e chemochine pro- ed anti-infiammatorie prodotte da cellule immunitarie, nonché da glia e microglia a livello dei nervi, dei gangli della radice dorsale (DRG) e del midollo spinale sono coinvolte nell’insorgenza e nel mantenimento del dolore neuropatico (Sacerdote et al., 2013). Tra le chemochine, la famiglia delle prochineticine, ha un ruolo importante nell'immunomodulazione, nell'infiammazione, nella nocicezione, nel dolore infiammatorio e in diversi modelli sperimentali di dolore neuropatico come quello indotto da lesione del nervo sciatico (Lattanzi et al., 2015; Guida et al., 2015) o da diabete (Castelli et al., 2016). Sia nell'uomo che nei roditori, la famiglia delle Prochineticine è composta da due proteine, la Prochineticina-1 (PK1) e la Prochineticina-2 (PK2) (Negri et al., 2007), che legano due recettori accoppiati a proteine G, PK-R1 e PK-R2 (Masuda et al., 2002). Le prochineticine ed i loro recettori sono ampiamente distribuiti nel cervello, nel midollo spinale, nei gangli della radice dorsale, nei granulociti, nei macrofagi e nei linfociti. È stato dimostrato che la PK2 è in grado di indurre un profilo pro-infiammatorio nei macrofagi (Martucci et al., 2006), che a sua volta può portare ad una riduzione delle soglie nocicettive in risposta sia a stimoli termici che meccanici (Negri et al., 2006; Giannini et al., 2009; Maftei et al., 2014). E’ stata dimostrata una co-localizzazione dei recettori PK-Rs e TRPV in alcune regioni nervose associate al dolore, ed è stato inoltre evidenziato che se stimolati con la PK2 (in vitro) molti neuroni che esprimono i recettori PK sono in grado di rilasciare mediatori del dolore come CGRP e sostanza P (Vellani et al., 2006). Sulla base di queste considerazioni, il presente progetto ha lo scopo di studiare il ruolo del sistema delle prochineticine in un modello murino di CIPN indotto dalla somministrazione di due diversi agenti chemioterapici comunemente usati in oncologia e correlati allo sviluppo di CIPN: vincristina (VCR), il farmaco più neurotossico tra gli alcaloidi della Vinca e bortezomib (BTZ), il primo inibitore del proteasoma 26s. Questi farmaci, nonostante abbiano meccanismi d’azione diversi sulle cellule tumorali, inducono neurotossicità e conseguente neuropatia attraverso meccanismi molto simili (Flatters et al., 2019; Boyette-Davis et al., 2015). Tutti gli esperimenti sono stati condotti utilizzando topi maschi del ceppo C57BL/6J di 9 settimane di età. Sulla base dei protocolli più comunemente utilizzati in letteratura, la CIPN è stata indotta negli animali attraverso la somministrazione intraperitoneale (i.p.) di bortezomib 0,4 mg/kg, 3 volte a settimana per 4 settimane consecutive (Boehmerle et al., 2014); mentre la vincristina è stata somministrata quotidianamente per 14 giorni consecutivi alla dose di 0,1 mg/kg (i.p.) (Kiguchi et al., 2008). Gli animali controllo sono stati trattati con il veicolo. Poichè l'allodinia è uno dei sintomi più comunemente riscontrati sia nei pazienti che nei modelli sperimentali di CIPN, l'allodinia meccanica e termica sono state monitorate durante gli esperimenti, utilizzando rispettivamente il Von Frey test e il test dell’acetone. Inoltre, è stata monitorata l'iperalgesia termica mediante il plantar test. La somministrazione di bortezomib o di vincristina ha indotto negli animali una condizione neuropatica dolorosa caratterizzata dalla presenza di allodinia meccanica, termica e di iperalgesia termica. Per valutare il possibile effetto terapeutico di PC1, un antagonista non peptidico dei PK-Rs, gli animali trattati con BTZ o VCR, in un chiaro stato di ipersensibilità, sono stati sottoposti al trattamento cronico con PC1 (150μg/kg, iniettato per via sottocutanea, due volte al giorno per 14 (BTZ) o 7 (VCR) giorni consecutivi). Indipendentemente dall'agente chemioterapico utilizzato per indurre la neuropatia, il trattamento cronico con PC1 è stato in grado di migliorare significativamente la sintomatologia dolorosa. Per valutare nel nostro modello (BTZ e VCR), il ruolo del sistema PK, le analisi biochimiche sono state eseguite a due diversi tempi sperimentali: prima dell’inizio del trattamento cronico con PC1 (rispettivamente 14 o 7 giorni dalla prima somministrazione di bortezomib e vincristina) e al termine del protocollo sperimentale (28 o 14 giorni dalla prima somministrazione con BTZ e VCR). Quattordici giorni dopo la prima somministrazione di BTZ, non sono state osservate alterazioni dell'espressione del sistema PK nelle principali stazioni coinvolte nella trasmissione del dolore (nervo sciatico, DRG e midollo spinale). Gli animali trattati con VCR invece, 7 giorni dopo la prima somministrazione di chemioterapico, mostravano, sia a livello del sistema nervoso periferico (DRG) che del sistema nervoso centrale (midollo spinale), un’up-regolazione di entrambi recettori del sistema PK. È noto dalla letteratura che il reclutamento e l'infiltrazione dei macrofagi a livello dei DRG svolge un ruolo chiave nello sviluppo e nel mantenimento della CIPN (Lees et al., 2017). Inoltre, è stato dimostrato che la PK2 è in grado di indurre i macrofagi a migrare e acquisire un fenotipo pro-infiammatorio, abbiamo quindi deciso di valutare l'espressione dei markers del macrofago/microglia (CD68 e CD11b) e del TLR4, la cui attivazione è correlata alla modulazione delle citochine pro-infiammatorie, nei tessuti nervosi dei nostri animali. A metà del protocollo chemioterapico (14 giorni BTZ e 7 giorni VCR), la sintomatologia dolorosa era correlata ad un aumento dell'espressione di CD68 e del TLR4. Inoltre, negli animali BTZ, le analisi di immunofluorescenza hanno confermato la presenza di macrofagi attivati sia a nel DRG che nel nervo sciatico. Allo stesso tempo (14 giorni BTZ e 7 giorni VCR), nei tessuti nervosi periferici di tutti gli animali sottoposti a trattamento con i chemioterapici, abbiamo osservato un profilo pro-infiammatorio caratterizzato da alti livelli delle citochine pro-infiammatorie IL-1β e IL-6 e bassi livelli dell’ anti-infiammatoria IL-10. A questi tempi sperimentali, il sistema nervoso centrale non sembrava essere alterato dalla neurotossicità della chemioterapia. Al termine del protocollo sperimentale (BTZ giorno 28 e VCR giorno 14), nel nervo sciatico e nei DRG degli animali BTZ, è stato rilevato un profilo neuroinfiammatorio più marcato; l'aumentata produzione di citochine potrebbe essere stata sostenuta dall'infiltrazione di macrofagi attivati. Infatti, negli animali con CIPN, a livello del nervo sciatico e dei DRG abbiamo osservato un up-regolazione del CD68, del CD11b e del TLR4 oltre che un aumento delle cellule CD68 positive nei topi BTZ. Queste cellule esprimevano anche PK2, suggerendo che i macrofagi infiltranti potrebbero rappresentare una fonte di PK2 nei tessuti nervosi periferici. Allo stesso tempo (BTZ giorno 28 e VCR giorno 14), anche il midollo spinale è stato influenzato dalla neurotossicità correlata alla chemioterapia; era infatti evidente un’upregolazione di tutti i componenti del sistema PK, del CD68 e del TLR4 oltre ad un chiaro profilo pro-infiammatorio. Inoltre, è stata osservata una colocalizzazione tra PK2 e GFAP (marker astrocitario) a livello del midollo spinale degli animali BTZ, suggerendo che, in questa stazione nervosa, gli astrociti potrebbero essere tra i principali produttori di PK2. Anche negli animali VCR, a livello del sistema nervoso centrale, era presente un aumento dell’espressione del GFAP e del CD68. PC1 è stato in grado di contrastare l'attivazione immunitaria e l'amplificazione neuroinfiammatoria, il cui ruolo nel mantenimento dell'ipersensibilità è noto. Infatti, il trattamento con l'antagonista ha portato ad una riduzione dell'espressione del CD68, del CD11b e del TLR4 e ha ristabilito un corretto equilibrio delle citochine sia a livello del sistema nervoso periferico che centrale. Poiché nella pratica clinica molti pazienti sono sottoposti a multipli cicli di chemioterapia (Starobova & Vetter, 2017), gli animali precedentemente trattati con gli agenti chemioterapici sono stati sottoposti ad un secondo ciclo BTZ o VCR. Dopo l'interruzione del primo ciclo di chemioterapia, gli animali hanno progressivamente recuperato le loro soglie di risposta basali. In entrambi i protocolli (BTZ e VCR), il secondo ciclo di chemioterapia ha indotto nuovamente lo sviuluppo di ipersensibilità negli animali precedentemente trattati con lo stesso agente chemioterapico. L'effetto allodinico indotto dai sia da BTZ che da VCR era meno evidente nei topi che erano stati precedentemente trattati anche con PC1 rispetto a quello osservato negli animali trattati in entrambi i cicli con il solo chemioterapico, indicando un possibile ruolo protettivo di PC1. Nella seconda parte del nostro studio, considerando la forte suscettibilità del DRG alla tossicità dei chemioterapici (Montague e Malcangio, 2017), abbiamo iniziato a studiare il coinvolgimento dei neuroni nella CIPN usando un modello in vitro rappresentato da neuroni sensitivi primari di DRG. In particolare, ci siamo concentrati sulla crescita dei neuriti, un fenotipo morfologico delle cellule neuronali correlato alla loro funzione ed al loro stato di salute (da Silva e Dotti, 2002). I neuroni prelevati da topi wildtype sono stati messi in coltura e sono state testate diverse concentrazioni di BTZ e VCR (Alè et al., 2015). Entrambi gli agenti hanno indotto una riduzione dose-dipendente della lunghezza totale dei neuriti rispetto alle cellule incubate con il solo veicolo. Basandoci sulla letteratura, abbiamo selezionato diverse concentrazioni di PC1 (Severini et al., 2015) che sono state utilizzate in associazione a BTZ e VCR. La dose più efficace di PC1 nel prevenire la tossicità indotta dai chemioterapici si è dimostrata essere la 250 nM. Abbiamo quindi iniziato ad analizzare i parametri biochimici delle cellule trattate con vincristina da sola o in associazione con PC1. Le cellule VCR hanno mostrato un upregolazione di: PK2, PK-R1, TLR4, IL-1β, IL-6, IL-10 e ATF3; che non era rilevabile nelle cellule co-trattate con VCR e PC1. VCR non ha indotto alterazioni dei livelli di mRNA di PK-R2, GFAP e TNF-α. Quando somministrato in assenza di vincristina, PC1 250nM non ha indotto alterazioni né nella morfologia cellulare né nei livelli di espressione dei parametri sopra citati. Nel loro insieme i nostri risultati mostrano che il sistema PK è coinvolto nello sviluppo e nel mantenimento della CIPN e, considerando gli effetti positivi del PC1 nel contrastare sia la sintomatologia dolorosa che la neuroinfiammazione, l'antagonismo dei PK-Rs potrebbe rappresentare un nuovo approccio terapeutico per gestire questo tipo di neuropatia dolorosa.
TARGETING PROKINETICIN SYSTEM TO COUNTERACT EXPERIMENTAL CHEMOTHERAPY-INDUCED PERIPHERAL NEUROPATHY / G. Moschetti ; tutore: P. Sacerdote ; coordinatore scuola di dottorato: A. L. Catapano. DIPARTIMENTO DI SCIENZE FARMACOLOGICHE E BIOMOLECOLARI, 2019 Dec 11. 32. ciclo, Anno Accademico 2019. [10.13130/moschetti-giorgia_phd2019-12-11].
TARGETING PROKINETICIN SYSTEM TO COUNTERACT EXPERIMENTAL CHEMOTHERAPY-INDUCED PERIPHERAL NEUROPATHY.
G. Moschetti
2019
Abstract
Chemotherapy induced peripheral neuropathy (CIPN) is a dose-limiting side effect of several antineoplastic agents commonly used in clinics. Up to now its management with common analgesic drugs is often unsatisfactory, therefore novel therapeutic approaches are needed. The molecular mechanisms underlying CIPN are still largely unknown, even if involvement of mitochondrial toxicity and oxidative stress, ion channels alterations and neuroinflammatory processes have been suggested (Boyette-Davis et al., 2015; Montague and Malcangio 2017). In particular, some cytokines and chemokines are involved in the initiation and persistence of pain (Sacerdote et al., 2013). Among chemokines, the Prokineticin family has an important role in immunomodulation, inflammation, nociception, inflammatory pain and in different experimental neuropathic conditions such as nerve lesion (Lattanzi et al., 2015; Guida et al., 2015) and diabetic neuropathy (Castelli et al., 2016). Both in humans and rodents, Prokineticin-1 (PK1) and Prokineticin-2 (PK2) (Negri et al., 2007) are ligands of two metabotropic receptors known as PK-R1 and PK-R2 (Masuda et al., 2002). Prokineticins and their receptors are expressed in the brain, spinal cord, dorsal root ganglia, granulocytes, macrophages and lymphocytes. It has been demonstrated that PK2 acts on macrophages and induces a pro-inflammatory profile (Martucci et al., 2006), which can lead to reduced nociceptive thresholds to thermal and mechanical stimuli (Negri et al., 2006; Giannini et al., 2009). Furthermore, in some nervous regions associated to pain, a co-localization of PK-Rs and TRPV receptors has been shown, and many neurons that respond to PK2 express and release typical pain mediators such as CGRP and substance P (Vellani et al., 2006). On the basis of these considerations, our study investigates the role of prokineticin system in a murine model of CIPN induced by the administration of two different chemotherapeutic agents commonly used in oncology and related to CIPN development: Vincristine (VCR), the most neurotoxic drug among Vinca Alkaloids, and bortezomib (BTZ), the first approved 26s proteasome inhibitor. These drugs, despite having different mechanisms of action on tumor cells, induce neurotoxicity and consequent neuropathy with similar mechanisms (Flatters et al., 2019; Boyette-Davis et al., 2015). C57BL/6J, 9 weeks old, male mice were used in all the experiments. Based on the most common protocols used in literature, CIPN was induced in mice through the intraperitoneal (i.p.) administration of bortezomib 0.4mg/kg, 3 times a week for 4 consecutive weeks (Boehmerle et al., 2014). Vincristine was administered at the dose of 0.1mg/kg, i.p. injected daily for 14 consecutive days (Kiguchi et al., 2008). Control mice were injected with the vehicle. Allodynia is one of the most frequent symptoms of CIPN, therefore, mechanical and thermal allodynia were monitored using Von Frey and Acetone Drop Test, respectively. Furthermore, thermal hyperalgesia was evaluated by Plantar test. Both bortezomib and vincristine induced in mice a painful neuropathy characterized by the presence of mechanical and thermal allodynia as well as thermal hyperalgesia. To assess the potential therapeutic effect of PC1, a non-peptidic antagonist of PK-Rs, BTZ- and VCR- mice in a clear hypersensitivity condition were subjected to chronic treatment with PC1 (150μg/kg, s.c. injected, daily administered for 14 or 7 consecutive days in the BTZ and VCR protocol respectively). Independently from the chemotherapeutic agent used to induce CIPN, PC1 was effective in ameliorating the painful symptoms. To assess the role of PK system in our model, for each experimental protocol (BTZ and VCR) we performed biochemical analysis at two different time points: before starting PC1 treatment (BTZ day 14 and VCR day 7), and at the end of chemotherapeutic schedule (BTZ day 28 and VCR day 14). Fourteen days after the first BTZ administration (BTZ day 14), no alterations were detected in PK system expression in the main stations involved in pain transmission (sciatic nerve, DRG, spinal cord). On the other hand, VCR-mice at day 7 (VCR day 7) already showed an upregulation of both PK-Rs at the level of the peripheral (DRG) and central (spinal cord) nervous system. Recruitment and infiltration of macrophages in DRG is known to have a key role in CIPN (Lees et al., 2017) and since PK2 is able to induce the macrophage to migrate and to acquire a pro-inflammatory phenotype, we evaluated expression of the macrophages/microglia markers (CD68 and CD11b) and of TLR4, whose activation is linked to pro-inflammatory cytokines modulation. At BTZ day 14 as well as VCR day 7, painful symptoms were related to an increase of CD68 and TLR4 expression. Immunofluorescence analysis confirmed the presence of activated macrophages in DRG and sciatic nerve of BTZ-mice. At the same time points, both BTZ- and VCR- mice peripheral nervous tissues showed a pro-inflammatory profile with high levels of TNF-α, IL-1β and IL-6 and low levels of IL-10. At BTZ day 14 and VCR day 7, the central nervous system was not directly affected by chemotherapy. At the end of the chemotherapeutic schedule (BTZ day 28 and VCR day 14), a stronger neuroinflammatory profile was detected in sciatic nerves and DRG of mice undergoing chemotherapy. The increased cytokine production could be sustained by infiltrating macrophages since we detected in both nerve and DRG of BTZ- and VCR- treated mice also an upregulation of CD68, CD11b and TLR4 and an increase of CD68 positive cells in BTZ-mice. These cells also expressed PK2, suggesting that infiltrating macrophages could be a source of PK2 in peripheral nervous tissues. At the same time point (BTZ day 28 and VCR day 14), also spinal cord was affected by chemotherapy-related neurotoxicity. In fact, an upregulation of all PK system components, high levels of CD68 and TLR4 and a clear pro-inflammatory cytokine profile were evident. Moreover, a colocalization between PK2 and the astrocytes marker GFAP in the spinal cord of BTZ-mice was observed, suggesting that astrocytes could be the main producers of the PK2 in this nervous station. An upregulation of both GFAP and CD68 was also present in the central nervous system of VCR treated mice. PC1 was able to counteract immune/neuroinflammatory activation and amplification, which is important to enhance hypersensitivity. In fact, the antagonist prevented or reduced the expression of CD68, CD11b and of TLR4 and restored a correct cytokine balance both in the peripheral and central nervous system. To more closely resemble the clinical situation, where patients are often subjected to several cycle of chemotherapy (Starobova & Vetter, 2017), mice previously treated with the chemotherapeutic agents, were subjected to a second BTZ- or VCR cycle. Following the interruption of chemotherapy, mice progressively recovered from neuropathy. In both protocols the second chemotherapy cycle induced again hypersensitivity, but the allodynic effect induced by the chemotherapeutic drugs was less evident in mice previously treated with PC1 if compared to that observed in BTZ/VCR-only re-treated mice, indicating a possible protective role of PC1. In the second part of our study, considering the susceptibility of DRG to chemotherapy (Montague and Malcangio, 2017), we began to study the involvement of neurons in CIPN using an in vitro model represented by DRG primary sensory neurons. In particular, we focused on neurite outgrowth, a morphological phenotype of neuronal cells that correlates with their function and health (da Silva et al., 2002). Neurons collected from wildtype mice were cultured and different concentrations of bortezomib and vincristine were tested (Alè et al., 2015). Both agents induced a dose-related reduction of neurite length compared to vehicle-treated cells. Different concentrations of PC1, based on the literature, (Severini et al., 2015) were co-applied to BTZ and VCR. PC1 dose-dependently prevented chemotherapy induced toxicity and the most effective dose of the antagonist was 250nM. We started to analyse biochemical parameters of neurons exposed to VCR alone or in association to PC1. VCR-cells showed an upregulation of PK2 and PK-R1, TLR4, IL-1β, IL-6, IL-10, and ATF3, which was not detectable in VCR-cells exposed also to PC1. VCR did not induce alterations in PK-R2, GFAP and TNF-α mRNA levels. PC1 250nM alone was tested in the cell cultures: no morphological/biochemical alteration was detected. Taken together our results show that PK system is involved in the development and maintenance of CIPN and, considering the positive effects of PC1 in contrasting both painful symptomatology and neuroinflammation, PK-Rs antagonism could represent a new therapeutic approach to handle this type of neuropathy.File | Dimensione | Formato | |
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