Gregory J. Michael

Axotomy results in major changes in BDNF expression by dorsal root ganglion cells: BDNF expression in large trkB and trkC cells, in pericellular baskets, and in projections to deep dorsal horn and dorsal column nuclei.

Brain derived neurotrophic factor (BDNF) is normally expressed by a small number of predominantly trkA‐expressing dorsal root ganglion cells. Using immunocytochemistry and in situ hybridization, we have examined the effect of sciatic nerve section on the expression of BDNF in the adult rat. Following axotomy there was a long lasting (4‐week) increase in BDNF mRNA and protein in large‐diameter, trkB‐ and trkC‐expressing dorsal root ganglion cells. By 2 days postaxotomy, expression of BDNF mRNA had increased from 2% of trkB cells to 50%, and from 18% of trkC cells to 56%. In contrast, BDNF expression in most trkA cells was unchanged, although was increased in the small population of medium‐ and large‐sized trkA cells. Following axotomy, BDNF‐immunoreactive terminals appeared in the central axonal projections of large‐diameter cells, including the deep dorsal horn and gracile nucleus. Neuropeptide Y was also upregulated following axotomy and was coexpressed with BDNF in the cell bodies and central terminals of the large cells. Ultrastructural analysis in lamina IV of the spinal cord revealed that BDNF terminals in these central projections establish synaptic contacts. Immunoreactivity at 4 weeks was also observed in pericellular baskets that contained calcitonin gene‐related peptide (CGRP) and surrounded trkA‐ and trkB‐expressing cells in L4 and L5 lumbar ganglia. These baskets are likely to arise from local, highly immunoreactive, BDNF/CGRP/trkA‐expressing cells. Our results identify several novel targets for BDNF and imply that it acts locally in both autocrine and paracrine modes, as well as centrally in a synaptic mode, to modulate the response of somatosensory pathways in nerve injury.

Localisation of cannabinoid receptor 1 in rat dorsal root ganglion using in situ hybridisation and immunohistochemistry

In this study we used in situ hybridisation and double-labelling immunohistochemistry to characterise cannabinoid receptor 1 (CB(1)) expression in rat lumbar dorsal root ganglion (DRG) neurons.Approximately 25% of DRG neurons expressed CB(1) mRNA and displayed immunoreactivity for CB(1). Sixty-nine percent to 82% of CB(1)-expressing cells were also immunoreactive for neurofilament 200, indicative of myelinated A-fibre neurons, which tend to be large- and medium-sized DRG neurons (>600 microm(2)). Approximately 10% of CB1-expressing cells also expressed transient receptor potential vanilloid family ion channel 2 (TRPV2), the noxious heat-transducing channel found in medium to large lightly myelinated Adelta-fibre DRG neurons. Seventeen percent to 26% of CB(1)-expressing cells co-stained using Isolectin B4, 9-10% for calcitonin gene-related peptide and 11-20% for transient receptor potential vanilloid family ion channel 1 (TRPV1), predominantly markers of small non-myelinated C-fibre DRG neurons (<600 microm(2)). These findings suggest that whilst a wide range of DRG neuron phenotypes express CB(1), it is predominantly associated with myelinated fibres.

Omega-3 fatty acids reverse age-related decreases in nuclear receptors and increase neurogenesis in old rats.

Retinoic acid receptors (RARs), retinoid X receptors (RXRs), and peroxisome proliferator‐activated receptors (PPARs) are transcription factors involved in many cellular processes, such as learning and memory. RAR and RXR mRNA levels decrease with ageing, and the decreases can be reversed by retinoic acid treatment, which also alleviates age‐related memory deficits. The omega‐3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have neuroprotective effects in the aged brain and are endogenous ligands of RXR and PPAR. We investigated whether dietary EPA and DHA supplementation reverses age‐related declines in protein levels of these receptors in rat forebrain. Two studies were conducted comparing adult and old rats. In the first, old rats were fed standard or EPA/DHA‐enriched (270 mg/kg/day, EPA to DHA ratio 1.5:1) diets for 12 weeks. Analysis by Western blot revealed significant decreases in RARα, RXRα, RXRβ, and PPARγ in the forebrain with ageing, which were reversed by supplementation. Immunohistochemical analysis of the hippocampus showed significant age‐related decreases in RARα and RXRβ expression in CA1 and the dentate gyrus, which were restored by supplementation. Decreases in hippocampal doublecortin expression were also partially alleviated, suggesting a positive effect on neurogenesis. We also investigated the effects of DHA supplementation (300 mg/kg/day for 12 weeks) on RARα, RXRα, and RXRβ expression in the prefrontal cortex, striatum, and hippocampus. Overall, DHA supplementation appeared to increase receptor expression compared with the untreated old group. These observations illustrate additional mechanisms that might underlie the neuroprotective effects of omega‐3 fatty acids in ageing.

NGF and GDNF ameliorate the increase in ATF3 expression which occurs in dorsal root ganglion cells in response to peripheral nerve injury

Activating transcription factor‐3 (ATF3) is a member of the ATF/CREB transcription factor superfamily and is induced in dorsal root ganglion (DRG) cells after nerve injury. In order to study the regulation of ATF3, we have examined the effect of nerve growth factor (NGF) and glial cell line‐derived neurotrophic factor (GDNF) on ATF3 expression. In untreated rats, sciatic nerve transection induced ATF3 immunoreactivity in 82% of L4 DRG cells at 14 days after axotomy. Intrathecal delivery of NGF or GDNF for 2 weeks commencing immediately after injury reduced the ATF3 expression to 35 and 23% of DRG cells, respectively. Cell size analysis indicated that NGF had protected a population of mainly small‐ to medium‐sized cells, but that the GDNF had protected a population of both small and large cells. This effect was confirmed by double labelling for P2X3, CGRP and 200 kDa neurofilament, markers for small peptide‐poor cells, peptide‐rich cells and large cells, respectively. Thus GDNF reduced the percentage of ATF3‐immunoreactive P2X3 cells from 70 to 4%, and the percentage of ATF3‐immunoreactive neurofilament cells from 63 to 24%. NGF was less effective than GDNF in reducing ATF3 expression in these cell types, but reduced the percentage of ATF3‐immunoreactive CGRP cells from 10% to < 1%. These results show that ATF3 expression in specific populations of DRG cells can be modulated by exogenous supplementation of specific trophic factors, and suggest that ATF3 expression may normally be induced by the loss of target‐derived NGF and GDNF.

Immunohistochemical co-localization of transient receptor potential vanilloid (TRPV)1 and sensory neuropeptides in the guinea-pig respiratory system

Electrophysiological studies within the lung have documented the presence of heterogenous groups of afferent fibers composed of Adelta and C-fibers and studies of somatosensory nerves within the skin reveal a complex pattern of distribution of sensory neuropeptides and transient receptor potential vanilloid (TRPV)1 positive nerves. However, the anatomical location of these different subpopulations of nerves within the lung has not been extensively studied. In the present study we have demonstrated that TRPV1 axons represented only a small proportion of the total number of PGP9.5 staining nerves within guinea-pig tracheal epithelium and only half the number of TRPV1 axons was immunopositive for substance P. In contrast, most TRPV1 positive neurones found within guinea-pig intrapulmonary airways were found to co-localize with sensory neuropeptides substance P and calcitonin gene-related peptide within and beneath the epithelium, around blood vessels, within airway smooth muscle and alveoli, indicative of heterogeneity of TRPV1 positive axons throughout the airways. However, in the smooth muscle layer of the trachea there was evidence of substance P and calcitonin gene-related peptide containing nerves that did not stain for TRPV1. We also demonstrated a complete loss of TRVP1 positive axons in the trachea and intrapulmonary airways and associated loss of bronchoconstriction induced by capsaicin, in animals chronically treated with capsaicin. However, some neuropeptide immunoreactive axons remained in the smooth muscle layer of capsaicin-treated animals which could represent the small subset of neuropeptide containing fibers which do not co-localize with TRPV1. We have provided evidence of heterogeneity of TRPV1 positive nerve fibers, including fibers characterized by lack of co-localization with neuropeptides in various regions of the airways and the existence of neuropeptide containing fibers that were not TRPV1 positive in guinea-pigs.

MicroRNA-155 negatively affects blood–brain barrier function during neuroinflammation

Blood–brain barrier (BBB) dysfunction is a hallmark of neurological conditions such as multiple sclerosis (MS) and stroke. However, the molecular mechanisms underlying neurovascular dysfunction during BBB breakdown remain elusive. MicroRNAs (miRNAs) have recently emerged as key regulators of pathogenic responses, although their role in central nervous system (CNS) microvascular disorders is largely unknown. We have identified miR‐155 as a critical miRNA in neuroinflammation at the BBB. miR‐155 is expressed at the neurovascular unit of individuals with MS and of mice with experimental autoimmune encephalomyelitis (EAE). In mice, loss of miR‐155 reduced CNS extravasation of systemic tracers, both in EAE and in an acute systemic inflammation model induced by lipopolysaccharide. In cultured human brain endothelium, miR‐155 was strongly and rapidly upregulated by inflammatory cytokines. miR‐155 up‐regulation mimicked cytokine‐induced alterations in junctional organization and permeability, whereas inhibition of endogenous miR‐155 partially prevented a cytokine‐induced increase in permeability. Furthermore, miR‐155 modulated brain endothelial barrier function by targeting not only cell–cell complex molecules such as annexin‐2 and claudin‐1, but also focal adhesion components such as DOCK‐1 and syntenin‐1. We propose that brain endothelial miR‐155 is a negative regulator of BBB function that may constitute a novel therapeutic target for CNS neuroinflammatory disorders.—Lopez‐Ramirez, M. A., Wu, D., Pryce, G., Simpson, J. E., Reijerkerk, A., King‐Robson, J., Kay, O, de Vries, H. E., Hirst, M. C., Sharrack, B., Baker D., Male, D. K., Michael, G. J., Romero, I. A. MicroRNA‐155 negatively affects blood–brain barrier function during neuroinflammation. FASEB J. 28, 2551–2565 (2014). www.fasebj.org

Minocycline treatment inhibits microglial activation and alters spinal levels of endocannabinoids in a rat model of neuropathic pain

Activation of spinal microglia contributes to aberrant pain responses associated with neuropathic pain states. Endocannabinoids (ECs) are present in the spinal cord, and inhibit nociceptive processing; levels of ECs may be altered by microglia which modulate the turnover of endocannabinoids in vitro. Here, we investigate the effect of minocycline, an inhibitor of activated microglia, on levels of the endocannabinoids anandamide and 2-arachidonoylglycerol (2-AG), and the related compound N-palmitoylethanolamine (PEA), in neuropathic spinal cord. Selective spinal nerve ligation (SNL) in rats resulted in mechanical allodynia and the presence of activated microglia in the ipsilateral spinal cord. Chronic daily treatment with minocycline (30 mg/kg, ip for 14 days) significantly reduced the development of mechanical allodynia at days 5, 10 and 14 post-SNL surgery, compared to vehicle-treated SNL rats (P < 0.001). Minocycline treatment also significantly attenuated OX-42 immunoreactivity, a marker of activated microglia, in the ipsilateral (P < 0.001) and contralateral (P < 0.01) spinal cord of SNL rats, compared to vehicle controls. Minocycline treatment significantly (P < 0.01) decreased levels of 2-AG and significantly (P < 0.01) increased levels of PEA in the ipsilateral spinal cord of SNL rats, compared to the contralateral spinal cord. Thus, activation of microglia affects spinal levels of endocannabinoids and related compounds in neuropathic pain states.

Reactive oxygen species, dietary restriction and neurotrophic factors in age-related loss of myenteric neurons

We have studied the mechanisms underlying nonpathological age‐related neuronal cell death. Fifty per cent of neurons in the rat enteric nervous system are lost between 12 and 18 months of age in ad libitum (AL) fed rats. Caloric restriction (CR) protects almost entirely against this neuron loss. Using the ROS‐sensitive dyes, dihydrorhodamine (DHR) and 2‐[6‐(4′‐hydroxy)phenoxy‐3H‐xanthen‐3‐on‐9‐yl]benzoic acid (HPF) in vitro, we show that the onset of cell death is linked with elevated intraneuronal levels of reactive oxygen species (ROS). Treatment with the neurotrophic factors NT3 and GDNF enhances neuronal antioxidant defence in CR rats at 12–15 months and 24 months but not in adult or aged AL‐fed animals. To examine the link between elevated ROS and neuronal cell death, we assessed apoptotic cell death following in vitro treatment with the redox‐cycling drug, menadione. Menadione fails to increase apoptosis in 6‐month neurons. However, in 12–15mAL fed rats, when age‐related cell death begins, menadione induces a 7‐ to 15‐fold increase in the proportion of apoptotic neurons. CR protects age‐matched neurons against ROS‐induced apoptosis. Treatment with neurotrophic factors, in particular GDNF, rescues neurons from menadione‐induced cell death, but only in 12–15mCR animals. We hypothesize that CR enhances antioxidant defence through neurotrophic factor signalling, thereby reducing age‐related increases in neuronal ROS levels and in ROS‐induced cell death.

Dietary enrichment with omega-3 polyunsaturated fatty acids reverses age-related decreases in the GluR2 and NR2B glutamate receptor subunits in rat forebrain

Ageing is associated with a decrease in the brain content of omega-3 polyunsaturated fatty acids (PUFA), such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), and with decreased neuroplasticity. The glutamate receptor subunits GluR2 and NR2B play a significant role in forebrain synaptic plasticity. We investigated GluR2 and NR2B in the aged prefrontal cortex, hippocampus and striatum, and tested if treatment with a preparation containing EPA and DHA can reverse age-related changes. The study compared adult and old (3-4 and 24-26 month) rats, and the latter were fed a standard diet or a diet supplemented for 12 weeks with omega-3 PUFA at 270mg/kg/day (ratio EPA to DHA 1.5:1). Ageing was associated with decreases in the GluR2 and NR2B subunits in all structures. These decreases were fully reversed by omega-3 PUFA supplementation. Age-related changes in the phospholipid PUFA content were also seen. Decreases in DHA were mostly corrected by supplementation. This study supports the neuroprotective effect of omega-3 fatty acids in brain ageing, and illustrates specific mechanisms underlying this effect.

Nerve growth factor modulates the activation status and fast axonal transport of ERK 1/2 in adult nociceptive neurones

Mature dorsal root ganglion cells respond to neurotrophins, and the intracellular signalling pathways activated by neurotrophins have been characterized in vitro. We have now used immunocytochemistry and Western blots to examine the expression and activation of extracellular signal-regulated protein kinase-1/2 (ERK) in rat dorsal root ganglion cells in vivo, using antisera to total (tERK) and phosphorylated (pERK) forms. This has revealed a number of novel findings. tERK immunoreactivity is present in most dorsal root ganglion cells but is expressed most strongly in small (nociceptive) cells and, surprisingly, is absent in a population of large cells that expressed trkB or trkC but mainly lack p75(NTR) immunoreactivity. In contrast pERK is prominent in a few trkA cells and in satellite glial cells, and is further increased by NGF treatment. tERK and pERK both undergo fast anterograde and retrograde axonal transport, indicated by accumulation at a sciatic nerve ligature, and NGF reduces the level of retrograde pERK transport.