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Real-time PCR was performed in the 7500 Fast Real-Time PCR System (Life Technologies) using the fast Sybrgreen programme and expression levels of the indicated genes were calculated using the Ct method by the appropriate function of the software using actin as calibrator

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This effect might be particularly important in the clinical management of PDN, given the close association between neuropathic pain and sleep disturbances [26]

Changes in tryptophan metabolism, particularly shifts in kynurenine and related metabolites, may disrupt serotonergic pathways and mood regulation, while alterations in homocysteine levels may impair dopaminergic neurotransmission