Supplementary Material for: Intranasal application of diluted saline alleviates ischemic brain injury in association with suppression of vasopressin neurons

Introduction: Cerebral swelling and brain injury in ischemic stroke are closely related to increased vasopressin (VP) secretion. How to alleviate ischemic brain injury by suppressing VP hypersecretion through simply available approaches remains to be established. Methods: Using a rat model of middle cerebral artery occlusion (MCAO), testing effects of intranasal application of 0.09% NaCl (IAL) on brain damages, VP neuronal activity, synaptic inputs, astrocytic plasticity, olfactory bulb (OB) activity in immunohistochemistry, patch-clamp recording, Western blotting and co-immunoprecipitation. Results: IAL reduced MCAO-evoked neurological disorders, brain swelling, injury and loss of neurons, increase in c-Fos expression and excitation of supraoptic VP neurons. The effects of IAL on VP neurons were associated with its suppression of MCAO-evoked increase in the frequency of excitatory synaptic inputs and decrease in the expression of glial fibrillary acidic protein (GFAP) filaments around VP neurons. MCAO and IAL also caused similar but weaker reactions in putative oxytocin neurons. In the OB, MCAO also increased the firing rate of mitral cells in the lesioned side, which was reduced by IAL. Direct hypotonic challenge of OB slices increased the expression of glutamine synthetase and GFAP filaments in the glomerular bodies while reducing the firing rate of mitral cells. Blocking aquaporin 4 activity in the supraoptic and paraventricular nuclei in the MCAO side blocked MCAO-evoked VP increase and brain damages. Conclusion: IAL reduces ischemic stroke-evoked brain injury in association with suppressing VP neuronal activity through reducing excitatory synaptic inputs and astrocytic process retraction, which likely result from reducing mitral cell activation.