Spinal cord vascular degeneration impairs duloxetine penetration.

Introduction: Chronic pain is a prevalent physically debilitating health-related morbidity. Frontline analgesics are inadequate, providing only partial pain relief in only a proportion of the patient cohort. Here, we explore whether alterations in spinal cord vascular perfusion are a factor in reducing the analgesic capability of the noradrenaline reuptake inhibitor, duloxetine. Method: An established rodent model of spinal cord vascular degeneration was used. Endothelial-specific vascular endothelial growth factor receptor 2 knockout mouse was induced via hydroxytamoxifen administered via intrathecal injection. Duloxetine was administered via intraperitoneal injection, and nociceptive behavioural testing was performed in both WT and VEGFR2KO mice. LC-MS/MS was performed to explore the accumulation of duloxetine in the spinal cord in WT and VEGFR2KO mice. Results: Spinal cord vascular degeneration leads to heat hypersensitivity and a decline in capillary perfusion. The integrity of noradrenergic projections (dopa - hydroxylase labelled) in the dorsal horn remained unaltered in WT and VEGFR2KO mice. There was an association between dorsal horn blood flow with the abundance of accumulated duloxetine in the spinal cord and analgesic capacity. In VEGFR2KO mice, the abundance of duloxetine in the lumbar spinal cord was reduced and was correlated with reduced anti-nociceptive capability of duloxetine. Discussion: Here, we show that an impaired vascular network in the spinal cord impairs the anti-nociceptive action of duloxetine. This highlights that the spinal cord vascular network is crucial to maintaining the efficacy of analgesics to provide pain relief.

Diabetes-induced microvascular complications at the level of the spinal cord: a contributing factor in diabetic neuropathic pain.

KEY POINTS: Diabetes is thought to induce neuropathic pain through activation of dorsal horn sensory neurons in the spinal cord. Here we explore the impact of hyperglycaemia on the blood supply supporting the spinal cord and chronic pain development. In streptozotocin-induced diabetic rats, neuropathic pain is accompanied by a decline in microvascular integrity in the dorsal horn. Hyperglycaemia-induced degeneration of the endothelium in the dorsal horn was associated with a loss in vascular endothelial growth factor (VEGF)-A165 b expression. VEGF-A165 b treatment prevented diabetic neuropathic pain and degeneration of the endothelium in the spinal cord. Using an endothelial-specific VEGFR2 knockout transgenic mouse model, the loss of endothelial VEGFR2 signalling led to a decline in vascular integrity in the dorsal horn and the development of hyperalgesia in VEGFR2 knockout mice. This highlights that vascular degeneration in the spinal cord could be a previously unidentified factor in the development of diabetic neuropathic pain. ABSTRACT: Abnormalities of neurovascular interactions within the CNS of diabetic patients is associated with the onset of many neurological disease states. However, to date, the link between the neurovascular network within the spinal cord and regulation of nociception has not been investigated despite neuropathic pain being common in diabetes. We hypothesised that hyperglycaemia-induced endothelial degeneration in the spinal cord, due to suppression of vascular endothelial growth factor (VEGF)-A/VEGFR2 signalling, induces diabetic neuropathic pain. Nociceptive pain behaviour was investigated in a chemically induced model of type 1 diabetes (streptozotocin induced, insulin supplemented; either vehicle or VEGF-A165 b treated) and an inducible endothelial knockdown of VEGFR2 (tamoxifen induced). Diabetic animals developed mechanical allodynia and heat hyperalgesia. This was associated with a reduction in the number of blood vessels and reduction in Evans blue extravasation in the lumbar spinal cord of diabetic animals versus age-matched controls. Endothelial markers occludin, CD31 and VE-cadherin were downregulated in the spinal cord of the diabetic group versus controls, and there was a concurrent reduction of VEGF-A165 b expression. In diabetic animals, VEGF-A165 b treatment (biweekly i.p., 20 ng g-1 ) restored normal Evans blue extravasation and prevented vascular degeneration, diabetes-induced central neuron activation and neuropathic pain. Inducible knockdown of VEGFR2 (tamoxifen treated Tie2CreERT2 -vegfr2flfl mice) led to a reduction in blood vessel network volume in the lumbar spinal cord and development of heat hyperalgesia. These findings indicate that hyperglycaemia leads to a reduction in the VEGF-A/VEGFR2 signalling cascade, resulting in endothelial dysfunction in the spinal cord, which could be an undiscovered contributing factor to diabetic neuropathic pain.

Cancer Chemotherapy in Early Life Significantly Alters the Maturation of Pain Processing.

Advances in pediatric cancer treatment have led to a ten year survival rate greater than 75%. Platinum-based chemotherapies (e.g. cisplatin) induce peripheral sensory neuropathy in adult and pediatric cancer patients. The period from birth through to adulthood represents a period of maturation within nociceptive systems. Here we investigated how cisplatin impacts upon postnatal maturation of nociceptive systems. Neonatal Wistar rats (Postnatal day (P) 7) were injected (i.p.) daily with either vehicle (PBS) or cisplatin (1mg/kg) for five consecutive days. Neither group developed mechanical or thermal hypersensitivity immediately during or after treatment. At P22 the cisplatin group developed mechanical (P < 0.05) and thermal (P < 0.0001) hypersensitivity versus vehicle group. Total DRG or dorsal horn neuronal number did not differ at P45, however there was an increase in intraepidermal nerve fiber density in cisplatin-treated animals at this age. The percentage of IB4+ve, CGRP+ve and NF200+ve DRG neurons was not different between groups at P45. There was an increase in TrkA+ve DRG neurons in the cisplatin group at P45, in addition to increased TrkA, NF200 and vGLUT2 immunoreactivity in the lumbar dorsal horn versus controls. These data highlight the impact pediatric cancer chemotherapy has upon the maturation of pain pathways and later life pain experience.

Identification of mechano-sensitive C fibre sensitization and contribution to nerve injury-induced mechanical hyperalgesia.

BACKGROUND: C fibre hyperexcitability is fundamental to chronic pain development in humans and rodents; therefore, peripheral sensory neuronal sensitization plays a role in the development of mechanical hyperalgesia. However, the axonal properties and underlying mechanisms that are associated to these chronic pain states still require investigation. METHODS: Teased fibre electrophysiology of the saphenous nerve was used to identify C fibres in naïve and nerve-injured rats. C fibres were identified using electrical stimulation which further provided conduction velocity slowing profiles. From these nerve filaments evoked responses to mechanical stimuli were recorded. Vehicle or galanin were applied directly to the saphenous nerve trunk prior to stimulation. RESULTS: Increased levels of mechanically evoked activity in mechano-sensitive C fibres was associated to reduced conduction failure, enhanced conduction velocity latency recovery and reduced conduction velocity slowing. Mechanical hyperalgesia developed in nerve-injured animals in which mechano-sensitive C fibres demonstrated increased mechanically evoked responses and reduced rate of adaptation. Mechano-sensitive C fibres in nerve-injured animals had reduced levels of conduction velocity slowing, enhanced rate of conduction velocity recovery and reduced firing frequency failure versus naïve animals; all hallmarks of enhanced sensory neuronal excitability. Directly applying the antinociceptive agent galanin to the saphenous nerve trunk in naive animals led to increased conduction failure, reduced latency recovery rate and increased levels of conduction velocity slowing. DISCUSSION: Nerve injury-induced enhanced neural responses to mechanical stimulation are associated to defined parameters setout by conduction velocity slowing, mediated via axonal processing. Application of galanin inhibits axonal excitability.

Regulation of alternative VEGF-A mRNA splicing is a therapeutic target for analgesia.

Vascular endothelial growth factor-A (VEGF-A) is best known as a key regulator of the formation of new blood vessels. Neutralization of VEGF-A with anti-VEGF therapy e.g. bevacizumab, can be painful, and this is hypothesized to result from a loss of VEGF-A-mediated neuroprotection. The multiple vegf-a gene products consist of two alternatively spliced families, typified by VEGF-A165a and VEGF-A165b (both contain 165 amino acids), both of which are neuroprotective. Under pathological conditions, such as in inflammation and cancer, the pro-angiogenic VEGF-A165a is upregulated and predominates over the VEGF-A165b isoform. We show here that in rats and mice VEGF-A165a and VEGF-A165b have opposing effects on pain, and that blocking the proximal splicing event - leading to the preferential expression of VEGF-A165b over VEGF165a - prevents pain in vivo. VEGF-A165a sensitizes peripheral nociceptive neurons through actions on VEGFR2 and a TRPV1-dependent mechanism, thus enhancing nociceptive signaling. VEGF-A165b blocks the effect of VEGF-A165a. After nerve injury, the endogenous balance of VEGF-A isoforms switches to greater expression of VEGF-Axxxa compared to VEGF-Axxxb, through an SRPK1-dependent pre-mRNA splicing mechanism. Pharmacological inhibition of SRPK1 after traumatic nerve injury selectively reduced VEGF-Axxxa expression and reversed associated neuropathic pain. Exogenous VEGF-A165b also ameliorated neuropathic pain. We conclude that the relative levels of alternatively spliced VEGF-A isoforms are critical for pain modulation under both normal conditions and in sensory neuropathy. Altering VEGF-Axxxa/VEGF-Axxxb balance by targeting alternative RNA splicing may be a new analgesic strategy.

Endogenous analgesic action of the pontospinal noradrenergic system spatially restricts and temporally delays the progression of neuropathic pain following tibial nerve injury.

Pontospinal noradrenergic neurons form part of an endogenous analgesic system that suppresses acute pain, but there is conflicting evidence about its role in neuropathic pain. We investigated the chronology of descending noradrenergic control during the development of a neuropathic pain phenotype in rats following tibial nerve transection (TNT). A lumbar intrathecal cannula was implanted at the time of nerve injury allowing administration of selective α-adrenoceptor (α-AR) antagonists to sequentially assay their effects upon the expression of allodynia and hyperalgesia. Following TNT animals progressively developed mechanical and cold allodynia (by day 10) and subsequently heat hypersensitivity (day 17). Blockade of α2-AR with intrathecal yohimbine (30 µg) revealed earlier ipsilateral sensitization of all modalities while prazosin (30 µg, α1-AR) was without effect. Established allodynia (by day 21) was partly reversed by the re-uptake inhibitor reboxetine (5 µg, i.t.) but yohimbine no longer had any sensitising effect. This loss of effect coincided with a reduction in the descending noradrenergic innervation of the ipsilateral lumbar dorsal horn. Yohimbine reversibly unmasked contralateral hindlimb allodynia and hyperalgesia of all modalities and increased dorsal horn c-fos expression to an innocuous brush stimulus. Contralateral thermal hyperalgesia was also reversibly uncovered by yohimbine administration in a contact heat ramp paradigm in anaesthetised TNT rats. Following TNT there is an engagement of inhibitory α2-AR-mediated noradrenergic tone which completely masks contralateral and transiently suppresses the development of ipsilateral sensitization. This endogenous analgesic system plays a key role in shaping the spatial and temporal expression of the neuropathic pain phenotype after nerve injury.