Nerve growth factor gene therapy improves bone marrow sensory innervation and nociceptor-mediated stem cell release in a mouse model of type 1 diabetes with limb ischaemia

AIMS/HYPOTHESIS: Sensory neuropathy is common in people with diabetes; neuropathy can also affect the bone marrow of individuals with type 2 diabetes. However, no information exists on the state of bone marrow sensory innervation in type 1 diabetes. Sensory neurons are trophically dependent on nerve growth factor (NGF) for their survival. The aim of this investigation was twofold: (1) to determine if sensory neuropathy affects the bone marrow in a mouse model of type 1 diabetes, with consequences for stem cell liberation after tissue injury; and (2) to verify if a single systemic injection of the NGF gene exerts long-term beneficial effects on these phenomena. METHODS: A mouse model of type 1 diabetes was generated in CD1 mice by administration of streptozotocin; vehicle was administered to non-diabetic control animals. Diabetic animals were randomised to receive systemic gene therapy with either human NGF or β-galactosidase. After 13 weeks, limb ischaemia was induced in both groups to study the recovery post injury. When the animals were killed, samples of tissue and peripheral blood were taken to assess stem cell mobilisation and homing, levels of substance P and muscle vascularisation. An in vitro cellular model was adopted to verify signalling downstream to human NGF and related neurotrophic or pro-apoptotic effects. Normally distributed variables were compared between groups using the unpaired Student’s t test and non-normally distributed variables were assessed by the Wilcoxon–Mann–Whitney test. The Fisher’s exact test was employed for categorical variables. RESULTS: Immunohistochemistry indicated a 3.3-fold reduction in the number of substance P-positive nociceptive fibres in the bone marrow of type 1 diabetic mice (p < 0.001 vs non-diabetic). Moreover, diabetes abrogated the creation of a neurokinin gradient which, in non-diabetic mice, favoured the mobilisation and homing of bone-marrow-derived stem cells expressing the substance P receptor neurokinin 1 receptor (NK1R). Pre-emptive gene therapy with NGF prevented bone marrow denervation, contrasting with the inhibitory effect of diabetes on the mobilisation of NK1R-expressing stem cells, and restored blood flow recovery from limb ischaemia. In vitro hNGF induced neurite outgrowth and exerted anti-apoptotic actions on rat PC12 cells exposed to high glucose via activation of the canonical neurotrophic tyrosine kinase receptor type 1 (TrkA) signalling pathway. CONCLUSIONS/INTERPRETATION: This study shows, for the first time, the occurrence of sensory neuropathy in the bone marrow of type 1 diabetic mice, which translates into an altered modulation of substance P and depressed release of substance P-responsive stem cells following ischaemia. NGF therapy improves bone marrow sensory innervation, with benefits for healing on the occurrence of peripheral ischaemia. Nociceptors may represent a new target for the treatment of ischaemic complications in diabetes. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s00125-019-4860-y) contains peer-reviewed but unedited supplementary material, which is available to authorised users.