On the histochemical labelling and quantitative stereological assessment of synaptic puncta within the dorsal horn: with a focus on synaptic remodelling following a lesion to the peripheral nerve

Peripheral nerve injury often results in neuropathic pain, which is chronic, maladaptive, and hard to treat. Accompanying the injury to the peripheral nerve are a series of changes within the central nervous system. Understanding these changes and how they relate to the neuropathic condition is vital in driving evidence-based treatment strategies. Neuronal connectivity is widely believed to underlie many cognitive processes, and altered connectivity may underlie the development and maintenance of neuropathic pain. Here, a tractable method of analysing connectivity, via the histological marking and fluorescence imaging of synaptic puncta, was developed. This method utilised automated image processing methods, and developed a stereological filter for unbiased object measurement. Using this approach, different synaptic markers were analysed in the superficial dorsal horn of rodents, a key component in the reception and processing of somatosensory information. Analysing PSD95 and SynaptoPhysin fluorescent puncta in naive adult rodents revealed distinct puncta distributions in the dorsal horn, reflecting gross circuit formation throughout this region. Peripheral nerve lesion to aspects of the sciatic nerve of rodents resulted in a selective loss at 21 days post injury of PSD95 puncta from lamina II inner of the superficial dorsal horn, with Synaptophysin puncta remaining unchanged. These experiments were complemented by a transgenic approach, where eGFP was expressed exclusively and permanently in sensory neurons, which also revealed no loss of sensory neuron afferent input to the dorsal horn after nerve lesion up to 21 days post injury. Finally, the hypothesis that microglial cells may be driving this synapse loss was explored by treating animals with minocycline following peripheral nerve lesion, which did not interfere with the PSD95 puncta loss observed at 21 days post injury.