Small Heat Shock Protein 27 Protects against Chemotherapy Induced Peripheral Neuropathy in Mice
Student thesis: Doctoral Thesis
Related Research Unit(s)
Patients receiving chemotherapy experience peripheral neuropathy at some stage of treatment. Taxol and vincristine are commonly used anti-neoplastic drugs. Cancer patients withdraw from their lifesaving chemotherapy because of its adverse effects. Chemotherapy-induced peripheral neuropathy (CIPN) is a major adverse effect, which causes unbearable neuropathic pain and affects the quality of life of patients. The major clinical symptom of CIPN is the development of mechanical and thermal/cold allodynia. Elucidating the mechanism underlying CIPN can enable its effective treatment or prevention. Taxol excessively polymerises microtubules, whereas vincristine depolymerises microtubules, resulting in the disruption of microtubule dynamics. This disruption results in inefficient axonal transport and thereby causes peripheral nerve degeneration. Several ion channel modulations, mitochondrial dysfunction, immune-activated inflammatory response and neuronal apoptosis are the major pathophysiological changes along with the axonal degeneration. These changes cause persistent painful peripheral neuropathy conditions. Our previous studies reported reduced axonal regeneration and alteration in the cytoskeleton of dorsal root ganglion (DRG) neurons after vincristine and Taxol treatments. DRG sensory neurons play a critical role in transducing pain signals. Heat shock proteins (HSPs) are inducible molecular chaperones, which protect sensory neurons from neurotoxicity. Heat shock protein 27 (Hsp27) is one of the first injury-induced genes identified in DRG neurons. In cultured DRG neurons, the expression of Hsp27 enhances neurite growth possibly because of its promoting action in actin polymerisation. In addition, the binding of Hsp27 with tubulin and microtubule increases the stability of the microtubule network in DRG neurons. Overexpression of human heat shock protein 27 (hHsp27) accelerates axonal regeneration with complete motor and sensory recovery in the peripheral nerve injury mouse model. Therefore, the present study intended to discover the in vivo protective effect of hHsp27 on a CIPN mouse model. We generated transgenic human heat shock protein 27 (hHsp27 Tg) mouse lines which highly express hHsp27 in both sensory and motor neurons. Taxol and vincristine were injected intraperitoneally to induce CIPN in hHsp27 Tg mice and their wild-type littermates. In another set of study, sciatic nerve crush was performed on mice two days after the last injection of chemotherapeutics (i.e. taxol or vincristine). Behavioural analysis was performed for mechanical and cold allodynia development in the CIPN mouse and for sensory and motor functional recovery in the peripheral nerve injury model along with electrophysiological studies to determine the compound muscle action potential, sensory nerve action potential and nerve conduction velocity. Post 30 days in the CIPN mouse model, immunostaining was performed on the hind paw skin for quantification of intra-epidermal nerve fibre density, on sciatic nerve transverse sections for quantification of the total number of axons and myelin basic protein expression and on the lateral plantar muscle for analysis of neuromuscular junctions. TUNEL assay was performed on DRG neuron sections, and DRG cleaved caspase-3 protein expression was estimated using western blot to study neuronal apoptosis. Transmission electron microscopy of the tibial nerve was performed to determine mitochondrial swelling in unmyelinated and myelinated axons, and quantitative PCR analysis on sciatic nerve RNA samples was performed for mitochondrial fusion and fission proteins to determine the degree of mitochondrial dysfunction. Toluidine blue staining on semi-thin tibial nerve transverse sections was performed to observe axonal changes. The behavioural, immunohistochemical, electrophysiological and morphometric evaluations in this study clearly suggest that hHsp27 overexpression can prevent, protect and cure CIPN in mouse and that Hsp27 is as a potential therapeutic candidate to treat CIPN.
- CIPN, Hsp27, Taxol, Vincristine, Sciatic nerve crush