Chemotherapy Induced Peripheral Neurotoxicity (CIPN) is the most prevalent neurological complication of anticancer therapy and it is predominantly a sensory and length-dependent neuropathy. Nowadays monitoring the CIPN onset and progression in treated patients represents an unmet clinical need and no serum biomarkers are available at the moment. Neurofilaments Light Chain (NfL) is specifically expressed in axons and it is released into the interstitial fluid and blood after axonal damage. Blood NfL concentration is useful for clinical diagnostic evaluation in neurological disorders such as Alzheimer’s and Huntington’s diseases, Amyotrophic Lateral Sclerosis and Multiple Sclerosis. A recent study indicated NfL as a marker of axonal damage in patients with Charcot Marie Tooth, an inherited neuropathy, reporting an increase of NfL concentration in serum. The aim of this project was to study the axonal damage and to investigate NfL as a specific axonal damage biomarker for CIPN onset and progression in preclinical models. Cisplatin (CDDP), paclitaxel (PTX) and vincristine (VCR), which belong respectively to the platinum, taxanes and vinca alkaloids classes of anticancer drugs, exert their toxic activity by targeting different structures of the peripheral nervous system: the axon and the neuronal cell body. In order to pursue the aim, two different protocols were performed. In the first study, female Wistar rats were divided in CTRL arm, which remained untreated, CDDP- (2 mg/kg i.p 2qwx4 ws) and PTX- (10 mg/kg i.v q7dx4 ws) treated groups. At the end of treatment, the axonal damage was evident in PTX-treated group: in particular, the morphometric and morphological analysis indicated nerve degeneration and fiber loss. The damage progressed from distal to proximal level of caudal nerve according to CIPN pathology. These observations were confirmed by the neurophysiological investigation (nerve conduction velocity and amplitude). Moreover, in the caudal nerves, immunoistochemical analysis indicated a consistent infiltration of macrophages. Instead, CDDP treated animals presented a milder axonopathy and the macrophages infiltration was absent in the nerves. Interestingly, the NF-Light assay (Quanterix) indicated an increase in serum NfL concentration in PTX treated animals compared to CTRL and CDDP groups, according to the major axonal damage. Given these interesting results, a second experiment using PTX- (10 mg/kg i.v q7dx4 ws) and VCR- (0.2 mg/kg i.v. q7dx4 ws) treated animals was performed. In particular, the aim of this second experiment was to assess a weekly time course in order to compare the progression of CIPN pathology and the serum NfL concentration. The analysis and sample collection were performed at different weekly time points and at the end of treatment. The axonal damage, detected by morphological and neurophysiological analysis, increased during the treatment in both groups but it was higher in PTX treated animals compared to VCR one from the second week of treatment. In addition, a more abundant macrophage infiltration was detected in caudal nerves of PTX- treated animals at each time point compared to VCR ones. Serum NfL concentration correlated with the progression of the damage in both groups but was higher in PTX treated one compared to CTRL and VCR groups according to the major axonal damage. In conclusion, our results demonstrated that NfL dosage may be used as an important serum biomarker for monitoring CIPN pathology and for an early detection in order to prevent severe damage.
La neurotossicità periferica indotta da chemioterapici (CIPN) è una tra le complicanze neurologiche più frequenti della terapia antitumorale ed è caratterizzata prevalentemente da una neuropatia di tipo sensitivo e lunghezza dipendente. Al giorno d'oggi il monitoraggio, l'insorgenza e la progressione di CIPN nei pazienti trattati rappresenta un'esigenza clinica. Non sono disponibili biomarker sierologici. I Neurofilamenti a catena leggera (NfL) sono espressi specificamente negli assoni e vengono rilasciati nel fluido interstiziale e nel sangue in seguito al danno assonale. La concentrazione di NfL nel sangue è risultata utile nella valutazione diagnostica di diversi disturbi neurologici del sistema nervoso centrale. Un recente studio ha indicato NfL come marker di danno assonale in pazienti con Charcot Marie Tooth. In questi pazienti è stato rilevato un aumento della concentrazione di NfL nel siero. Lo scopo di questo progetto è stato studiare il danno assonale e valutare il possibile ruolo dei NfL come biomarker di danno assonale per l'insorgenza e la progressione della CIPN in modelli preclinici. Cisplatino (CDDP), paclitaxel (PTX) e vincristina (VCR) esercitano la loro attività tossica traghettando diverse strutture del sistema nervoso periferico: l'assone e il corpo cellulare del neurone. Al fine di perseguire l'obiettivo, sono stati eseguiti due protocolli diversi. Nel primo studio, ratti femmine Wistar sono state divise in CTRL (non trattati), trattati con CDDP (2 mg / kg i.p 2qwx4 ws) e con PTX (10 mg / kg i.v q7dx4 ws). Alla fine del trattamento, il danno assonale è risultato evidente nel gruppo trattato con PTX: in particolare, l'analisi morfometrica e morfologica ha indicato una degenerazione assonale e una perdita di fibre. Il danno al nervo caudale è progredito dal livello distale a quello prossimale confermando la fisiopatologia della CIPN. Queste osservazioni sono state confermate dall'analisi neurofisiologica (velocità di conduzione nervosa e ampiezza). Inoltre, nei nervi caudali, l'analisi immunoistochimica ha indicato un’importante infiltrazione di macrofagi. Al contrario, gli animali trattati con CDDP mostrano una lieve assonopatia accompagnata dall'assenza di infiltrato macrofagico. L’ NF-Light assay (Quanterix) ha indicato un aumento nel siero della concentrazione di NfL negli animali trattati con PTX rispetto ai gruppi CTRL e CDDP. Questi dati correlano col danno assonale maggiore. In seguito è stato eseguito un secondo esperimento con animali trattati con PTX- (10 mg / kg i.v q7dx4 settimane) e VCR- (0,2 mg / kg i.v. q7dx4 settimane). In particolare, in questo secondo esperimento è stato svolto un time-course ai fini di correlare la progressione della CIPN e la concentrazione di NfL nel siero. Le analisi e la raccolta dei campioni sono state eseguite a diversi punti sperimentali e alla fine del trattamento. Il danno assonale, rilevato dall'analisi morfologica e neurofisiologica, è aumentato durante il trattamento in entrambi i gruppi, ma è risultato più elevato negli animali trattati con PTX rispetto a quelli trattati con VCR a partire dalla seconda settimana di trattamento. Inoltre, è stata rilevata un'infiltrazione di macrofagi più importante nei nervi caudali degli animali trattati con PTX ad ogni tempistica rispetto a quelli trattati con VCR. La concentrazione di NfL nel siero è risultata correlata con la progressione del danno in entrambi i gruppi, ma è risultata maggiore nel gruppo trattato con PTX rispetto ai gruppi CTRL e VCR. Questi dati correlano col danno assonale maggiore. In conclusione, i nostri risultati hanno dimostrato che il dosaggio di NfL può essere usato come importante biomarker nel siero per il monitoraggio della CIPN e per una diagnosi precoce al fine di prevenire gravi danni.
(2020). The role of Neurofilament Light Chain as a serum biomarker in chemotherapy-induced peripheral neurotoxicity rat models. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2020).
The role of Neurofilament Light Chain as a serum biomarker in chemotherapy-induced peripheral neurotoxicity rat models
FUMAGALLI, GIULIA
2020
Abstract
Chemotherapy Induced Peripheral Neurotoxicity (CIPN) is the most prevalent neurological complication of anticancer therapy and it is predominantly a sensory and length-dependent neuropathy. Nowadays monitoring the CIPN onset and progression in treated patients represents an unmet clinical need and no serum biomarkers are available at the moment. Neurofilaments Light Chain (NfL) is specifically expressed in axons and it is released into the interstitial fluid and blood after axonal damage. Blood NfL concentration is useful for clinical diagnostic evaluation in neurological disorders such as Alzheimer’s and Huntington’s diseases, Amyotrophic Lateral Sclerosis and Multiple Sclerosis. A recent study indicated NfL as a marker of axonal damage in patients with Charcot Marie Tooth, an inherited neuropathy, reporting an increase of NfL concentration in serum. The aim of this project was to study the axonal damage and to investigate NfL as a specific axonal damage biomarker for CIPN onset and progression in preclinical models. Cisplatin (CDDP), paclitaxel (PTX) and vincristine (VCR), which belong respectively to the platinum, taxanes and vinca alkaloids classes of anticancer drugs, exert their toxic activity by targeting different structures of the peripheral nervous system: the axon and the neuronal cell body. In order to pursue the aim, two different protocols were performed. In the first study, female Wistar rats were divided in CTRL arm, which remained untreated, CDDP- (2 mg/kg i.p 2qwx4 ws) and PTX- (10 mg/kg i.v q7dx4 ws) treated groups. At the end of treatment, the axonal damage was evident in PTX-treated group: in particular, the morphometric and morphological analysis indicated nerve degeneration and fiber loss. The damage progressed from distal to proximal level of caudal nerve according to CIPN pathology. These observations were confirmed by the neurophysiological investigation (nerve conduction velocity and amplitude). Moreover, in the caudal nerves, immunoistochemical analysis indicated a consistent infiltration of macrophages. Instead, CDDP treated animals presented a milder axonopathy and the macrophages infiltration was absent in the nerves. Interestingly, the NF-Light assay (Quanterix) indicated an increase in serum NfL concentration in PTX treated animals compared to CTRL and CDDP groups, according to the major axonal damage. Given these interesting results, a second experiment using PTX- (10 mg/kg i.v q7dx4 ws) and VCR- (0.2 mg/kg i.v. q7dx4 ws) treated animals was performed. In particular, the aim of this second experiment was to assess a weekly time course in order to compare the progression of CIPN pathology and the serum NfL concentration. The analysis and sample collection were performed at different weekly time points and at the end of treatment. The axonal damage, detected by morphological and neurophysiological analysis, increased during the treatment in both groups but it was higher in PTX treated animals compared to VCR one from the second week of treatment. In addition, a more abundant macrophage infiltration was detected in caudal nerves of PTX- treated animals at each time point compared to VCR ones. Serum NfL concentration correlated with the progression of the damage in both groups but was higher in PTX treated one compared to CTRL and VCR groups according to the major axonal damage. In conclusion, our results demonstrated that NfL dosage may be used as an important serum biomarker for monitoring CIPN pathology and for an early detection in order to prevent severe damage.File | Dimensione | Formato | |
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