Abbreviations used: CHO, Chinese hamster ovary; CPCCOEt, 7-hydroxyiminocyclopropan[b]chromen-1a-carboxylic acid ethyl ester; Cy TM 3, cyanin 3; DAPI, 4¢,6-diamidino-2-phenylindole dilactate; DHK, dihydrokainic acid; DHPG, (S)-3,5-dihydroxyphenylglycine; FBS, foetal bovine serum; GFAP, glial fibrillary acidic protein; GLAST, glutamateaspartate transporter; GLT, glutamate transporters; GS, glutamine synthetase; IPTG, isopropyl-b-D-thiogalactoside; L-SOS, L-serine O-sulphate potassium salt; LTHA, L-())-threo-3-hydroxyaspartic acid; mGlu5 receptor, type 5 metabotropic glutamate receptor; MPEP, 2-methyl-6-(phenylethynyl)-pyridine.

BackgroundMultipotent mesenchymal stem (stromal) cells (MSCs) have been credited with immunomodulative properties, supporting beneficial outcomes when transplanted into a variety of disease models involving inflammation. Potential mechanisms include the secretion of paracrine factors and the establishment of a neurotrophic microenvironment. To test the hypothesis that MSCs release soluble mediators that can attenuate local inflammation, we here analysed the influence of MSCs on the activation of microglia cells, as well as on inflammatory parameters and pain behaviour in a surgical rat model of neuropathic pain.MethodsWe focussed on an experimental model of partial sciatic nerve ligation (PSNL), characterised by a rapid and persistent inflammation in the dorsal lumbar spinal cord where sensory inputs from the sciatic nerve are processed. Via indwelling intrathecal catheters, MSCs were repetitively grafted into the intrathecal lumbar space. Animals were evaluated for mechanical and thermal hypersensitivity over a period of 21 days after PSNL. Afterwards, spinal cords were processed for immunohistochemical analysis of the microglial marker ionized calcium-binding adapter molecule 1 (Iba1) and quantification of inflammatory markers in ipsilateral dorsal horns. We hypothesised that injections on postsurgical days 2 to 4 would interfere with microglial activation, leading to a reduced production of pro-inflammatory cytokines and amelioration of pain behaviour.ResultsPSNL-induced mechanical allodynia or heat hyperalgesia were not influenced by MSC transplantation, and spinal cord inflammatory processes remained largely unaffected. Indeed, the early microglial response to PSNL characterised by increased Iba1 expression in the lumbar dorsal horn was not significantly altered and cytokine levels in the spinal cord at 21 days after surgery were similar to those found in vehicle-injected animals. Grafted MSCs were detected close to the pia mater, but were absent within the spinal cord parenchyma.ConclusionsWe conclude that intrathecal administration is not an appropriate route to deliver cells for treatment of acute spinal cord inflammation as it leads to entrapment of grafted cells within the pia mater. We propose that the early inflammatory response triggered by PSNL in the lumbar spinal cord failed to effectively recruit MSCs or was insufficient to disturb the tissue integrity so as to allow MSCs to penetrate the spinal cord parenchyma.

BackgroundNeuroinflammation and nitroxidative stress are implicated in the pathophysiology of neuropathic pain. In view of both processes, microglial and astroglial activation in the spinal dorsal horn play a predominant role. The present study investigated the severity of neuropathic pain and the degree of glial activation in an inflammatory- and nitroxidative-prone animal model.MethodsTransgenic rats expressing mutated superoxide dismutase 1 (hSOD1G93A) are classically used as a model for amyotrophic lateral sclerosis (ALS). Because of the associated inflammatory- and nitroxidative-prone properties, this model was used to study thermal and mechanical hypersensitivity following partial sciatic nerve ligation (PSNL). Next to pain hypersensitivity assessment, microglial and astroglial activation states were moreover characterized, as well as inflammatory marker gene expression and the glutamate clearance system.ResultsPSNL induced thermal and mechanical hypersensitivity in both wild-type (WT) and transgenic rats. However, the degree of thermal hypersensitivity was found to be exacerbated in transgenic rats while mechanical hypersensitivity was only slightly and not significantly increased. Microglial Iba1 expression was found to be increased in the ipsilateral dorsal horn of the lumbar spinal cord after PSNL but such Iba1 up-regulation was enhanced in transgenic rats as compared WT rats, both at 3 days and at 21 days after injury. Moreover, mRNA levels of Nox2, a key enzyme in microglial activation, but also of pro-inflammatory markers (IL-1β and TLR4) were not modified in WT ligated rats at 21 days after PSNL as compared to WT sham group while transgenic ligated rats showed up-regulated gene expression of these 3 targets. On the other hand, the PSNL-induced increase in GFAP immunoreactivity spreading that was evidenced in WT rats was unexpectedly found to be attenuated in transgenic ligated rats. Finally, GLT-1 gene expression and uptake activity were shown to be similar between WT sham and WT ligated rats at 21 days after injury, while both parameters were significantly increased in the ipsilateral dorsal region of the lumbar spinal cord of hSOD1G93A rats.ConclusionsTaken together, our findings show that exacerbated microglial activation and subsequent inflammatory and nitroxidative processes are associated with the severity of neuropathic pain symptoms.

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Prepro-vasoactive intestinal peptide (VIP) mRNA codes for two neuropeptides: VIP and peptide histidine isoleucine (PHI). Two VIP receptors, shared with a similar affinity by pituitary adenylate cyclase-activating polypeptide (PACAP), have been cloned: VPAC 1 and VPAC 2 . PHI binds to these receptors with a lower affinity. VPAC receptors are classically associated with a cAMP-dependent pathway, although other pathways, including calcium mobilization and protein kinase C activation have been described. We previously showed that intracerebral administration of the glutamate agonist ibotenate to postnatal day 5 mice induces white matter lesions mimicking human periventricular leukomalacia. In this model, coinjection of VIP protects against white matter lesions. This neuroprotection is independent from cAMP and is mediated by protein kinase C. Using this model, this study aimed to determine the receptor involved in VIP-induced neuroprotection. VIP effects were mimicked with a similar potency by VPAC 2 agonists and PHI but not by VPAC 1 agonists, PACAP 27, or PACAP 38. VIP neuroprotective effects were lost in mice lacking VPAC 2 receptor. In situ hybridization confirmed the presence of VPAC 2 mRNA in the postnatal day 5 white matter. When analyzed between embryonic life and adulthood, VIP-specific binding site density peaked at postnatal day 5. These data suggest that, in this model, VIP-induced neuroprotection is mediated by VPAC 2 receptors. The pharmacology of this VPAC 2 receptor seems unconventional because 1) PACAP does not mimic VIP effects, 2) PHI acts with a comparable potency, and 3) PACAP 27 modestly inhibited the VIP-specific binding, whereas for PHI or VIP, inhibition was complete.