Ping K. Yip

Chondroitinase ABC Promotes Sprouting of Intact and Injured Spinal Systems after Spinal Cord Injury

Chondroitin sulfate proteoglycans (CSPGs) are inhibitory extracellular matrix molecules that are upregulated after CNS injury. Degradation of CSPGs using the enzyme chondroitinase ABC (ChABC) can promote functional recovery after spinal cord injury. However, the mechanisms underlying this recovery are not clear. Here we investigated the effects of ChABC treatment on promoting plasticity within the spinal cord. We found robust sprouting of both injured (corticospinal) and intact (serotonergic) descending projections as well as uninjured primary afferents after a cervical dorsal column injury and ChABC treatment. Sprouting fibers were observed in aberrant locations in degenerating white matter proximal to the injury in regions where CSPGs had been degraded. Corticospinal and serotonergic sprouting fibers were also observed in spinal gray matter at and below the level of the lesion, indicating increased innervation in the terminal regions of descending projections important for locomotion. Spinal-injured animals treated with a vehicle solution showed no significant sprouting. Interestingly, ChABC treatment in uninjured animals did not induce sprouting in any system. Thus, both denervation and CSPG degradation were required to promote sprouting within the spinal cord. We also examined potential detrimental effects of ChABC-induced plasticity. However, although primary afferent sprouting was observed after lumbar dorsal column lesions and ChABC treatment, there was no increased connectivity of nociceptive neurons or development of mechanical allodynia or thermal hyperalgesia. Thus, CSPG digestion promotes robust sprouting of spinal projections in degenerating and denervated areas of the spinal cord; compensatory sprouting of descending systems could be a key mechanism underlying functional recovery.

Inhibition of spinal microglial cathepsin S for the reversal of neuropathic pain

A recent major conceptual advance has been the recognition of the importance of immune system–neuronal interactions in the modulation of brain function, one example of which is spinal pain processing in neuropathic states. Here, we report that in peripheral nerve-injured rats, the lysosomal cysteine protease cathepsin S (CatS) is critical for the maintenance of neuropathic pain and spinal microglia activation. After injury, CatS was exclusively expressed by activated microglia in the ipsilateral dorsal horn, where expression peaked at day 7, remaining high on day 14. Intrathecal delivery of an irreversible CatS inhibitor, morpholinurea-leucine-homophenylalanine-vinyl phenyl sulfone (LHVS), was antihyperalgesic and antiallodynic in neuropathic rats and attenuated spinal microglia activation. Consistent with a pronociceptive role of endogenous CatS, spinal intrathecal delivery of rat recombinant CatS (rrCatS) induced hyperalgesia and allodynia in naïve rats and activated p38 mitogen-activated protein kinase (MAPK) in spinal cord microglia. A bioinformatics approach revealed that the transmembrane chemokine fractalkine (FKN) is a potential substrate for CatS cleavage. We show that rrCatS incubation reduced the levels of cell-associated FKN in cultured sensory neurons and that a neutralizing antibody against FKN prevented both FKN- and CatS-induced allodynia, hyperalgesia, and p38 MAPK activation. Furthermore, rrCatS induced allodynia in wild-type but not CX3CR1-knockout mice. We suggest that under conditions of increased nociception, microglial CatS is responsible for the liberation of neuronal FKN, which stimulates p38 MAPK phosphorylation in microglia, thereby activating neurons via the release of pronociceptive mediators.

P2X2 knockout mice and P2X2/P2X3 double knockout mice reveal a role for the P2X2 receptor subunit in mediating multiple sensory effects of ATP

Extracellular ATP plays a role in nociceptive signalling and sensory regulation of visceral function through ionotropic receptors variably composed of P2X2 and P2X3 subunits. P2X2 and P2X3 subunits can form homomultimeric P2X2, homomultimeric P2X3, or heteromultimeric P2X2/3 receptors. However, the relative contribution of these receptor subtypes to afferent functions of ATP in vivo is poorly understood. Here we describe null mutant mice lacking the P2X2 receptor subunit (P2X2−/−) and double mutant mice lacking both P2X2 and P2X3 subunits (P2X2/P2X3Dbl−/−), and compare these with previously characterized P2X3−/− mice. In patch‐clamp studies, nodose, coeliac and superior cervical ganglia (SCG) neurones from wild‐type mice responded to ATP with sustained inward currents, while dorsal root ganglia (DRG) neurones gave predominantly transient currents. Sensory neurones from P2X2−/− mice responded to ATP with only transient inward currents, while sympathetic neurones had barely detectable responses. Neurones from P2X2/P2X3Dbl−/− mice had minimal to no response to ATP. These data indicate that P2X receptors on sensory and sympathetic ganglion neurones involve almost exclusively P2X2 and P2X3 subunits. P2X2−/− and P2X2/P2X3Dbl−/− mice had reduced pain‐related behaviours in response to intraplantar injection of formalin. Significantly, P2X3−/−, P2X2−/−, and P2X2/P2X3Dbl−/− mice had reduced urinary bladder reflexes and decreased pelvic afferent nerve activity in response to bladder distension. No deficits in a wide variety of CNS behavioural tests were observed in P2X2−/− mice. Taken together, these data extend our findings for P2X3−/− mice, and reveal an important contribution of heteromeric P2X2/3 receptors to nociceptive responses and mechanosensory transduction within the urinary bladder.

CCL2 is a key mediator of microglia activation in neuropathic pain states

While neuroimmune interactions are increasingly recognized as important in nociceptive processing, the nature and functional significance of these interactions is not well defined. There are multiple reports that the activation of spinal microglia is a critical event in the generation of neuropathic pain behaviors but the mediators of this activation remain disputed. Here we show that the chemokine CCL2, produced by both damaged and undamaged primary sensory neurons in neuropathic pain states in rats, is released in an activity dependent manner from the central terminals of these fibres. We also demonstrate that intraspinal CCL2 in naïve rats leads to activation of spinal microglia and neuropathic pain‐like behavior. An essential role for spinal CCL2 is demonstrated by the inhibition of neuropathic pain behavior and microglial activation by a specific neutralising antibody to CCL2 administered intrathecally. Thus, the neuronal expression of CCL2 provides a mechanism for immune activation, which in turn regulates the sensitivity of pain signaling systems in neuropathic pain states.

A diacylglycerol lipase-CB2 cannabinoid pathway regulates adult subventricular zone neurogenesis in an age-dependent manner

The subventricular zone (SVZ) is a major site of neurogenesis in the adult. We now show that ependymal and proliferating cells in the adult mouse SVZ express diacylglycerol lipases (DAGLs), enzymes that synthesise a CB1/CB2 cannabinoid receptor ligand. DAGL and CB2 antagonists inhibit the proliferation of cultured neural stem cells, and the proliferation of progenitor cells in young animals. Furthermore, CB2 agonists stimulate progenitor cell proliferation in vivo, with this effect being more pronounced in older animals. A similar response was seen with a fatty acid amide hydrolase (FAAH) inhibitor that limits degradation of endocannabinoids. The effects on proliferation were mirrored in changes in the number of neuroblasts migrating from the SVZ to the olfactory bulb (OB). In this context, CB2 antagonists reduced the number of newborn neurons appearing in the OB in the young adult animals while CB2 agonists stimulated this in older animals. These data identify CB2 receptor agonists and FAAH inhibitors as agents that can counteract the naturally observed decline in adult neurogenesis that is associated with ageing.

Release of BDNF and GABA in the dorsal horn of neuropathic rats

Damage to peripheral nerves is associated with changes in excitability and/or phenotype of primary afferent neurons as well as increased neuronal excitability (central sensitization) and reduced inhibitory tone in the dorsal horn. For instance, in dorsal root ganglia (DRG) brain derived neurotrophic factor (BDNF) is down‐regulated in small cells whilst de novo expressed in large diameter cells. In the dorsal horn, GABA content is decreased. In this study, in a dorsal horn, ‘with dorsal roots attached’ preparation obtained from spinal nerve lesioned Wistar rats, stimulation of ipsilateral dorsal roots at either A fibre or A + C fibre strength did not evoke release of BDNF. In separate experiments, activity‐induced release of GABA in the isolated dorsal horn of neuropathic rats was significantly reduced compared to release in sham operated rats. GABA release could be significantly restored following topical application of BDNF through the dorsal horn preparation. Finally, neuropathic rats developed thermal and mechanical hypersensitivity and thermal hyperalgesia was reduced by intrathecal injection of BDNF. We concluded that BDNF‐induced release of GABA could be a mechanism to explain the antinociceptive action of intrathecal BDNF in neuropathic animals. Furthermore, reduced availability of sensory neuron‐derived BDNF might contribute to the reduced GABAergic tone in the dorsal horn of neuropathic rats.

The Liberation of Fractalkine in the Dorsal Horn Requires Microglial Cathepsin S

Understanding of the sequence and nature of the events that govern neuron–microglia communication is critical for the discovery of new mechanisms and targets for chronic pain treatment. The neuronal chemokine fractalkine (FKN) and its microglial receptor CX3CR1 may mediate such a function in the dorsal horn of the spinal cord after cleavage of the extracellular domain of this transmembrane chemokine by a protease. Here we report that in neuropathic rat dorsal horn, with dorsal root-attached preparations, soluble FKN (sFKN) contents are increased in the superfusates collected after noxious-like electrical stimulation of ipsilateral primary afferent fibers. The increase of sFKN is prevented by morpholinurea-leucine-homophenylalanine-vinyl sulfone-phenyl (LHVS), an irreversible inhibitor of cathepsin S (CatS) whose proteolytic activity is also increased in the superfusates. The source of CatS activity is microglial cells activated by the peripheral nerve injury and secreting the enzyme, as a result of primary afferent fiber stimulation. Indeed, the acute activation of dorsal horn microglia by lipopolysaccharide results in increased CatS activity in the superfusates, followed by increased sFKN contents. Consistent with these observations ex vivo, the levels of both sFKN and CatS activity in CSF samples increased significantly after peripheral nerve injury, associated with spinal microglial activation. Finally, because we found that both FKN immunoreactivity and mRNA are confined to dorsal horn neurons, we suggest that under neuropathic conditions, noxious stimulation of primary afferent fibers induces release of CatS from microglia, which liberates FKN from dorsal horn neurons, thereby contributing to the amplification and maintenance of chronic pain.

Retinoic acid receptor |[beta]|2 promotes functional regeneration of sensory axons in the spinal cord

The embryonic CNS readily undergoes regeneration, unlike the adult CNS, which has limited axonal repair after injury. Here we tested the hypothesis that retinoic acid receptor β2 (RARβ2), critical in development for neuronal growth, may enable adult neurons to grow in an inhibitory environment. Overexpression of RARβ2 in adult rat dorsal root ganglion cultures increased intracellular levels of cyclic AMP and stimulated neurite outgrowth. Stable RARβ2 expression in DRG neurons in vitro and in vivo enabled their axons to regenerate across the inhibitory dorsal root entry zone and project into the gray matter of the spinal cord. The regenerated neurons enhanced second-order neuronal activity in the spinal cord, and RARβ2-treated rats showed highly significant improvement in sensorimotor tasks. These findings show that RARβ2 induces axonal regeneration programs within injured neurons and may thus offer new therapeutic opportunities for CNS regeneration.

Assessing behavioural function following a pyramidotomy lesion of the corticospinal tract in adult mice.

We have developed a pyramidotomy model in mice to lesion the corticospinal tract at the level of the brainstem pyramidal tract, and evaluated the resultant impairments in motor function in a series of behavioural tests. Adult C57BL/6 mice received a unilateral pyramidotomy and a control group of mice underwent sham surgery. We studied the effects of this lesion on forepaw function using five behavioural paradigms, some of which have been widely used in rat studies but have not been fully explored in mice. The tests used were: a rearing test, which assesses forepaw use for weight support during spontaneous vertical exploration of a cylinder; a grid walking test, which assesses the ability to accurately place the forepaws during exploration of an elevated grid; a tape-removal test, which measures both sensory and motor function of the forepaw; a CatWalk automated gait analysis, which provides a number of quantitative measures including stride length and stride width during locomotion; and a staircase reaching task, which assesses skilled independent forepaw use. All tests revealed lesion effects on forepaw function with the tape removal, grid walking, rearing and CatWalk tests demonstrating robust effects throughout the testing period. The development of a pyramidotomy lesion model in mice, together with behavioural tests which can reliably measure functional impairments, will provide a valuable tool for assessing therapeutic strategies to promote regeneration and plasticity.

Prolonged anticonvulsant action of glutamate metabotropic receptor agonists in inferior colliculus of genetically epilepsy-prone rats

The anticonvulsant activity of (S)-4-carboxy-3-hydroxyphenylglycine ((S)-4C3HPG) (an antagonist of Group I and an agonist of Group II metabotropic glutamate (mGlu) receptors), of (1S,3S)-1-aminocyclopentane-1,3-dicarboxylic acid ((1S,3S)-ACPD) (an agonist of Group II mGlu receptors), and of L-serine-O-phosphate (an agonist of Group III mGlu receptors) was studied against sound-induced seizures in genetically epilepsy-prone (GEP) rats following bilateral microinjection into the inferior colliculus. All 3 drugs produce dose-dependent suppression of all phases of sound-induced seizures (wild running, clonic and tonic). (S)-4C3HPG produces an immediate and short-lasting (< 2 h) protection against sound-induced seizures with an ED50 value of 4.3 (3.2-5.7) nmol, at 5 min. The preferential agonists of Group II and Group III mGlu receptors produce an immediate, transient (< 10 min) proconvulsant effect followed by a prolonged (> 1 day) anticonvulsant effect against sound-induced seizures. The anticonvulsant ED50 value for (1S,3S)-ACPD is 9 (5-18) nmol at 2 h, and for L-serine-O-phosphate is 36 (6.5-199) nmol at 2 days. It is concluded that mGlu receptor activation potently modifies seizure threshold.