Resumen de Grape stalk and coffee polyphenolic extracts administration as new pharmacological strategies to modulate spinal cord injury-induced neuropathic pain in mice
Central neuropathic pain (CNP) following spinal cord injury (SCI) is developed in more than half of patients and around one-third of those report the pain to be severe. CNP after SCI has been associated with impairments in a variety of areas, including physical functioning and mobility, mental and cognitive functioning, social functioning and community reintegration, sleep, employment, and quality of life. Moreover, people who experience pain after SCI show poorer health, lower life satisfaction and a higher risk of depression. To address this health concern, several pharmacological treatments have been used to alleviate CNP. However, current treatments are often ineffective because they target only one or two of the several mechanisms that comprise CNP. Nowadays, to design a personalized therapeutic approach, treatment consists in a trial-and-error of different strategies, including antidepressants, antiepileptics, GABA-agonists, local anaesthetics, NMDA-antagonists, cannabinoids and opioids. However, these drugs are usually inadequate and only one third of patients respond to pharmacological treatments when compared with placebo. Moreover, the best pharmacological strategy results in a reduction of only 20–30% in pain intensity, frequently accompanied by severe side effects. Hence, given the lack of effective treatments, it is necessary to develop new pharmacological strategies not only for the neuropathic pain relief but also for the prevention of its chronification. Among the potential pharmacological strategies aimed at modulating pathological pain could be highlighted the use of polyphenols, since preclinical evidence of their antinociceptive effects can be found in the scientific literature. The properties that can be attributed to polyphenols that may explain pathological pain modulation are free radical scavenging/antioxidant, immunomodulatory, neuroprotective, anti-apoptotic and autophagy-regulating activities. However, although several studies have been specifically aimed at elucidating the effects of polyphenolic treatments on the neuropathic pain development, most of them have been conducted in preclinical models unrelated to SCI, such as peripheral neuropathic pain, diabetic neuropathic pain, or alcoholic neuropathy, among others. Furthermore, although studies on polyphenol treatment after SCI are available, most of them have focused on motor recovery or spinal cord regeneration, leading to a lack of information despite promising results on their effects on modulating pathophysiological processes that may also be related to neuropathic pain development. In this context, the main objective of the present thesis was to study the preventive and analgesic effects of two polyphenolic plant extracts in SCI-induced CNP development in mice and to compare it with the effects of epigallocatechin-3-gallate (EGCG), one of the most studied polyphenols in the field of neuropathic pain. Concretely, the two polyphenolic extracts were obtained from grape residual material (GSE) and roasted decaffeinated coffee powder (CE), which were chosen since both grapevine and coffee are known to be rich natural sources of polyphenols. After SCI, the effects of repeated EGCG, GSE and CE treatment on neuropathic pain behaviours were evaluated during the acute, intermediate, and chronic phase of injury. In addition, gliosis and the expression central sensitisation-related biomarkers were analysed in both spinal cord and pain-processing involved supraspinal structures. On the one hand, the EGCG administration during the first week post SCI resulted in both mechanical allodynia and thermal hyperalgesia development prevention during the acute phase of SCI, accompanied by SCI-induced spinal cord gliosis attenuation. However, all tested doses of EGCG resulted in significant weight loss of mice throughout the experimental period, indicating that systemic toxicity could be associated with such treatment. In contrast, both preventive GSE and CE administration during the first week post SCI, resulted in the attenuation of both mechanical allodynia and thermal hyperalgesia development during the acute phase of SCI without either weight-loss or serum biomarkers increase of hepatotoxicity or nephrotoxicity. Antinociceptive effects of both polyphenolic extracts were associated with the prevention of both gliosis and upregulation of central sensitization-related algogens (pERK, CX3CL1, CX3CR1 and CCR2) in the spinal cord. In addition, while GSE and CE treatments modulated CX3CL1/CX3CR1 signalling and attenuated astrogliosis in both supraspinal structures of the anterior cingulate cortex (ACC) and periaqueductal gray (PAG), as well as microgliosis in the ACC of injured mice, such treatments induced microglial activation in PAG likely characterized by an increase of microglial cells exhibiting an M2 anti‐inflammatory phenotype. On the other hand, while repeated administration of EGCG during the third week post SCI modulated neither mechanical allodynia nor thermal hyperalgesia induced by mild SCI during the intermediate phase of injury, the same administration pattern for both GSE and CE modulated these reflexive pain responses with no associated systemic toxicity, hepatotoxic or nephrotoxic effects. In addition, CE administration modulated depressive-like behaviour detected in spinal cord injured mice at intermediate phase of injury. Finally, repeated administration of GSE and CE during the first-, third- and sixth-week post SCI prevented thermal hyperalgesia and mechanical allodynia development up to the chronic phase of injury, with no associated systemic toxicity, hepatotoxic or nephrotoxic effects. In addition, GSE and CE treatments also modulated both affective-motivational disturbances (anhedonia, depression and anxiety) and social interaction impairment developed in chronic SCI-animals. Antinociceptive effects of GSE and CE were associated not only with the modulation of spinal cord microgliosis but also with the modulation of astrogliosis and CCL2/CCR2 and CX3CL1/CX3CR1 signalling in the supraspinal structures ACC, amygdala, dorsal and ventral PAG and rostral ventromedial medulla (RVM) as well as microgliosis in ventral PAG. Overall, the results of the present thesis suggest that a mixture of polyphenols present in natural extracts may be a suitable pharmacological strategy to either prevent or attenuate the development of SCI-induced neuropathic pain by modulating not only the reflexive pain responses (more related to the sensory-discriminative dimension of pain) but also the non-reflexive pain responses (included in the affective-motivational dimension of pain). These compounds not only exert their effects at the site of injury by modulating gliosis and the expression of central sensitisation-related biomarkers but also on supraspinal structures closely related to expression and modulation of central neuropathic pain.
