Aim: Osteoarthritis (OA) is a disabling and painful condition very common in the elderly. Pain is the earliest symptom of OA. To date there are still no curative drugs for this condition. Moreover, the chronic use of first-line pharmacological treatments to handle OA pain is frequently associated with side effects. CR4056, an imidazoline-2 receptor ligand, is a promising analgesic drug that has been reported to be effective in several animal models of pain. The aims of my project were to analyze and compare the time-related progression of OA pain and to evaluate the efficacy of CR4056, in comparison with a standard NSAID (naproxen), in two well-established rat models of OA, able to mimic the painful and structural components of the human pathology. Methods: Knee OA was induced either by single intra-articular injection of 1 mg/50 μl monosodium iodoacetate (MIA) or by medial meniscal tear (MMT) in the right knee of male rats. The local injection of MIA produces cartilage degeneration, through the local inhibition of glycolisis, while the transection of both the medial collateral ligament and the medial meniscus leads to joint destabilization, resulting in cartilage degeneration and subchondral bone alterations. The withdrawal threshold to mechanical stimulation was assessed both as allodynia and either as primary or secondary hyperalgesia, in MIA and MMT model, respectively. Pain behaviour was further evaluated as static and dynamic hind paw weight bearing (HPWD) asymmetry between the ipsilateral and the contralateral limb, and as changes in motor function and/or locomotor activity. Pain-related proteins (GFAP, pp38, pERKs and Iba-1) expression was assessed in either ipsilateral and contralateral lumbar spinal cord or ipsilateral L4 and L5 dorsal root ganglions (DRGs), in either MIA or MMT model. CR4056 (2, 6 and 20 mg/kg) and 10 mg/kg naproxen were administered as acute and sub-acute treatments in both models. Results: MIA model was characterized by the significant development of primary mechanical hyperalgesia, mechanical allodynia and asymmetry in both static and dynamic HPWD. No changes were detected in locomotor activity after MIA injection. 6 and 20 mg/kg CR4056 significantly and dose-dependently reduced both allodynia and hyperalgesia, after acute (7 and 14 days after MIA) and especially after repeated treatment (from 14 to 21 days post-MIA), whereas naproxen was effective after sub-acute treatment only. Both compounds had no significant effect on static and dynamic HPWD changes. No difference was detected in pp38 and pERKs expression in ipsilateral lumbar spinal cord, between MIA and sham group. On the other hand, a significant increase in the number of Iba-1 positive, morphologically identified, activated microglia in ipsilateral L4 spinal cord dorsal horn occurred, 21 days after MIA injection. Sub-acute treatment with 6 mg/kg CR4056 and naproxen reversed MIA-induced microglia activation. MMT surgery induced the significant development of a progressive asymmetry in static HPWD and a long-lasting secondary mechanical hyperalgesia. No mechanical allodynia nor changes in dynamic HPWD, motor function and locomotor activity were detected after MMT surgery. 20 mg/kg CR4056 and naproxen promoted a mild but significant anti-hyperalgesic effect, after acute treatment (28 days post-surgery) only. Conversely, repeated treatment (from 28 to 42 days post-surgery) with 6 mg/kg CR4056 significantly reduced the progression of static HPWD asymmetry, whereas naproxen had no effects. No difference in GFAP or Iba-1 expression were detected, in either ipsilateral L4 and L5 DRGs or ipsilateral L4 spinal cord dorsal horn, between MMT and sham group. Conclusions: Both MIA and MMT OA models display a pain behaviour comparable to human OA, with however different relative contribution of peripheral and central pain mechanisms. Moreover, the data obtained showed that CR4056 may represent a new effective treatment option for OA pain.
L'osteoartrite (OA) è una patologia degenerativa cronica molto comune negli anziani. Il dolore rappresenta il sintomo di OA più invalidante e precoce per il paziente. Ad oggi non esistono terapie curative per questa patologia. Inoltre, i farmaci più comunemente usati per contrastare il dolore OA sono spesso associati ad effetti collaterali. CR4056, ligando dei recettori imidazolinici-2, è un nuovo farmaco analgesico dimostratosi efficace in diversi modelli animali di dolore. Gli scopi di questo progetto sono stati analizzare la progressione del dolore OA e valutare l'efficacia di CR4056, comparato ad un FANS standard (naproxene), in due modelli di OA in ratto, in grado di replicare le componenti dolorose e strutturali della patologia umana. L’OA è stata indotta mediante singola iniezione intra-articolare di 1 mg/50 μl di monosodio iodoacetato (MIA) o mediante rottura del menisco mediale (MMT) nel ginocchio destro di ratti maschi. L’iniezione di MIA causa la degenerazione della cartilagine, mediante inibizione locale della glicolisi, mentre la resezione del legamento collaterale mediale e del menisco mediale destabilizza l’articolazione, inducendo degradazione della cartilagine e alterazioni dell’osso subcondrale. Nei modelli MIA e MMT la soglia algogena meccanica è stata valutata come allodinia e rispettivamente iperalgesia primaria o secondaria. Sono stati poi determinati l’asimmetria statica o dinamica del peso supportato delle zampe posteriori ipsilaterale e contralaterale (HPWD), la funzionalità motoria e l’attività locomotoria. L’espressione di proteine correlate al dolore (GFAP, pp38, pERKs e Iba-1) è stata valutata nel midollo spinale lombare ipsilaterale e contralaterale o nei DRG L4 e L5 ipsilaterali. CR4056 (2, 6 e 20 mg/kg) e naproxene (10 mg/kg) sono stati somministrati come trattamenti acuti e subacuti in entrambi i modelli. L’iniezione di MIA ha indotto uno sviluppo significativo di iperalgesia meccanica primaria, allodinia meccanica e asimmetria statica e dinamica di HPWD; nessun cambiamento si è verificato nell’attività locomotoria. CR4056 (6 e 20 mg/kg) ha ridotto significativamente l’allodinia e l’iperalgesia, dopo trattamento acuto (7 e 14 giorni post MIA) e soprattutto subacuto (da 14 a 21 giorni post MIA), mentre il naproxene è stato efficace solo dopo trattamento subacuto. Entrambi i composti sono risultati privi di effetto sull’asimmetria statica e dinamica di HPWD. L’espressione di pp38 e pERKs nel midollo spinale lombare ipsilaterale non si è dimostrata differente tra sham e MIA. L’iniezione di MIA ha invece indotto un aumento significativo delle cellule microgliali attivate (Iba1+) nella corna dorsale ipsilaterale del midollo spinale L4. Il trattamento subacuto con entrambi i composti ha ridotto significativamente tale attivazione della microglia. L’operazione MMT ha indotto uno sviluppo significativo di una progressiva asimmetria statica di HPWD e di una stabile iperalgesia meccanica secondaria. Non sono stati invece evidenziati allodinia, asimmetria dinamica di HPWD e cambiamenti nell’attività locomotoria e funzionalità motoria. CR4056 (20 mg/kg) e naproxene hanno dimostrato un significativo effetto anti-iperalgesico solo dopo trattamento acuto (28 giorni post MMT). Invece, il trattamento subacuto (da 28 a 42 giorni post MMT) con CR4056 (6 mg/kg) ha ridotto significativamente l’asimmetria statica di HPWD, mentre il naproxene è risultato privo di effetti. Non si sono verificate variazioni nell’espressione di GFAP e Iba-1, rispettivamente nei DRG L4 e L5 ipsilaterali e nella corna dorsale ipsilaterale del midollo spinale L4, tra sham e MMT. Entrambi i modelli MIA e MMT sono caratterizzati da una componente di dolore OA comparabile a quella umana, con tuttavia un differente contributo dei meccanismi dolorosi periferici e centrali. Inoltre, i dati ottenuti dimostrano che CR4056 potrebbe costituire un nuovo efficace trattamento per il dolore OA.
(2017). Chronic pain evaluation in animal models of osteoarthritis: behavioural and pharmacological considerations.. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2017).
Chronic pain evaluation in animal models of osteoarthritis: behavioural and pharmacological considerations.
COMI, ELEONORA
2017
Abstract
Aim: Osteoarthritis (OA) is a disabling and painful condition very common in the elderly. Pain is the earliest symptom of OA. To date there are still no curative drugs for this condition. Moreover, the chronic use of first-line pharmacological treatments to handle OA pain is frequently associated with side effects. CR4056, an imidazoline-2 receptor ligand, is a promising analgesic drug that has been reported to be effective in several animal models of pain. The aims of my project were to analyze and compare the time-related progression of OA pain and to evaluate the efficacy of CR4056, in comparison with a standard NSAID (naproxen), in two well-established rat models of OA, able to mimic the painful and structural components of the human pathology. Methods: Knee OA was induced either by single intra-articular injection of 1 mg/50 μl monosodium iodoacetate (MIA) or by medial meniscal tear (MMT) in the right knee of male rats. The local injection of MIA produces cartilage degeneration, through the local inhibition of glycolisis, while the transection of both the medial collateral ligament and the medial meniscus leads to joint destabilization, resulting in cartilage degeneration and subchondral bone alterations. The withdrawal threshold to mechanical stimulation was assessed both as allodynia and either as primary or secondary hyperalgesia, in MIA and MMT model, respectively. Pain behaviour was further evaluated as static and dynamic hind paw weight bearing (HPWD) asymmetry between the ipsilateral and the contralateral limb, and as changes in motor function and/or locomotor activity. Pain-related proteins (GFAP, pp38, pERKs and Iba-1) expression was assessed in either ipsilateral and contralateral lumbar spinal cord or ipsilateral L4 and L5 dorsal root ganglions (DRGs), in either MIA or MMT model. CR4056 (2, 6 and 20 mg/kg) and 10 mg/kg naproxen were administered as acute and sub-acute treatments in both models. Results: MIA model was characterized by the significant development of primary mechanical hyperalgesia, mechanical allodynia and asymmetry in both static and dynamic HPWD. No changes were detected in locomotor activity after MIA injection. 6 and 20 mg/kg CR4056 significantly and dose-dependently reduced both allodynia and hyperalgesia, after acute (7 and 14 days after MIA) and especially after repeated treatment (from 14 to 21 days post-MIA), whereas naproxen was effective after sub-acute treatment only. Both compounds had no significant effect on static and dynamic HPWD changes. No difference was detected in pp38 and pERKs expression in ipsilateral lumbar spinal cord, between MIA and sham group. On the other hand, a significant increase in the number of Iba-1 positive, morphologically identified, activated microglia in ipsilateral L4 spinal cord dorsal horn occurred, 21 days after MIA injection. Sub-acute treatment with 6 mg/kg CR4056 and naproxen reversed MIA-induced microglia activation. MMT surgery induced the significant development of a progressive asymmetry in static HPWD and a long-lasting secondary mechanical hyperalgesia. No mechanical allodynia nor changes in dynamic HPWD, motor function and locomotor activity were detected after MMT surgery. 20 mg/kg CR4056 and naproxen promoted a mild but significant anti-hyperalgesic effect, after acute treatment (28 days post-surgery) only. Conversely, repeated treatment (from 28 to 42 days post-surgery) with 6 mg/kg CR4056 significantly reduced the progression of static HPWD asymmetry, whereas naproxen had no effects. No difference in GFAP or Iba-1 expression were detected, in either ipsilateral L4 and L5 DRGs or ipsilateral L4 spinal cord dorsal horn, between MMT and sham group. Conclusions: Both MIA and MMT OA models display a pain behaviour comparable to human OA, with however different relative contribution of peripheral and central pain mechanisms. Moreover, the data obtained showed that CR4056 may represent a new effective treatment option for OA pain.File | Dimensione | Formato | |
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phd_unimib_720663.pdf
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Descrizione: tesi di dottorato
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Doctoral thesis
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