Biomedical subjects
C J Woolf
Publications and source records attributed to C J Woolf.
Neuropathic pain: aetiology, symptoms, mechanisms, and management.
We highlight current theories about peripheral neuropathic pain and show that progress in management is contingent on targeting treatment not at the aetiological factors or the symptoms but at the mechanisms that operate to produce the symptoms. This approach will require substantial progress in our understanding of the pathophysiology of neuropathic pain, the development of accurate diagnostic tools to discover what mechanisms contribute to the pain syndrome in an individual, and effective treatments aimed specifically at the mechanisms.
Peripheral inflammation facilitates Abeta fiber-mediated synaptic input to the substantia gelatinosa of the adult rat spinal cord.
Whole-cell patch-clamp recordings were made from substantia gelatinosa (SG) neurons in thick adult rat transverse spinal cord slices with attached dorsal roots to study changes in fast synaptic transmission induced by peripheral inflammation. In slices from naive rats, primary afferent stimulation at Abeta fiber intensity elicited polysynaptic EPSCs in only 14 of 57 (25%) SG neurons. In contrast, Abeta fiber stimulation evoked polysynaptic EPSCs in 39 of 62 (63%) SG neurons recorded in slices from rats inflamed by an intraplantar injection of complete Freund's adjuvant (CFA) 48 hr earlier (p < 0.001). Although the peripheral inflammation had no significant effect on the threshold and conduction velocities of Abeta, Adelta, and C fibers recorded in dorsal roots, the mean threshold intensity for eliciting EPSCs was significantly lower in cells recorded from rats with inflammation (naive: 33.2 +/- 15.1 microA, n = 57; inflamed: 22.8 +/- 11.3 microA, n = 62, p < 0.001), and the mean latency of EPSCs elicited by Abeta fiber stimulation in CFA-treated rats was significantly shorter than that recorded from naive rats (3.3 +/- 1.8 msec, n = 36 vs 6.0 +/- 3.5 msec, n = 12; p = 0.010). Abeta fiber stimulation evoked polysynaptic IPSCs in 4 of 25 (16%) cells recorded from naive rat preparations and 14 of 26 (54%) SG neurons from CFA-treated rats (p < 0.001). The mean threshold intensity for IPSCs was also significantly lower in CFA-treated rats (naive: 32.5 +/- 15.7 microA, n = 25; inflamed: 21. 9 +/- 9.9 microA, n = 26, p = 0.013). The facilitation of Abeta fiber-mediated input into the substantia gelatinosa after peripheral inflammation may contribute to altered sensory processing.
Peripheral inflammation increases the capsaicin sensitivity of dorsal root ganglion neurons in a nerve growth factor-dependent manner.
Inflammation results in a local increase in nerve growth factor production which potentially can modify the properties of nerve growth factor-responsive sensory neurons innervating the inflamed tissue. The sensitivity of primary sensory neurons to the neurotoxin capsaicin is regulated in vitro by nerve growth factor and we have now investigated the effect of complete Freund's adjuvant-induced inflammation on the capsaicin sensitivity of adult rat sensory neurons. Dorsal root ganglion neurons innervating inflamed tissue were identified in vivo by retrograde labelling with the dye Fast Blue. Neuronal capsaicin sensitivity was measured in vitro with a quantitative cobalt-uptake densitometric technique, and was shown to increase significantly five days after inflammation. This increase in sensitivity was dependent on nerve growth factor as it could be inhibited by systemic treatment with nerve growth factor neutralizing antibodies. The enhanced capsaicin sensitivity that results from Freund's adjuvant injection may contribute to inflammatory hyperalgesia.
Regeneration of dorsal column fibers into and beyond the lesion site following adult spinal cord injury.
Regeneration is abortive following adult mammalian CNS injury. We have investigated whether increasing the intrinsic growth state of primary sensory neurons by a conditioning peripheral nerve lesion increases regrowth of their central axons. After dorsal column lesions, all fibers stop at the injury site. Animals with a peripheral axotomy concomitant with the central lesion show axonal growth into the lesion but not into the spinal cord above the lesion. A preconditioning lesion 1 or 2 weeks prior to the dorsal column injury results in growth into the spinal cord above the lesion. In vitro, the growth capacity of DRG neurite is also increased following preconditioning lesions. The intrinsic growth state of injured neurons is, therefore, a key determinant for central regeneration.
Nociceptive-specific activation of ERK in spinal neurons contributes to pain hypersensitivity.
We investigated the involvement of extracellular signal-regulated protein kinases (ERK) within spinal neurons in producing pain hypersensitivity. Within a minute of an intense noxious peripheral or C-fiber electrical stimulus, many phosphoERK-positive neurons were observed, most predominantly in lamina I and IIo of the ipsilateral dorsal horn. This staining was intensity and NMDA receptor dependent. Low-intensity stimuli or A-fiber input had no effect. Inhibition of ERK phosphorylation by a MEK inhibitor reduced the second phase of formalin-induced pain behavior, a measure of spinal neuron sensitization. ERK signaling within the spinal cord is therefore involved in generating pain hypersensitivity. Because of its rapid activation, this effect probably involves regulation of neuronal excitability without changes in transcription.
Heat shock protein 27: developmental regulation and expression after peripheral nerve injury.
The heat shock protein (HSP) 27 is constitutively expressed at low levels in medium-sized lumbar dorsal root ganglion (DRG) cells in adult rats. Transection of the sciatic nerve results in a ninefold upregulation of HSP27 mRNA and protein in axotomized neurons in the ipsilateral DRG at 48 hr, without equivalent changes in the mRNAs encoding HSP56, HSP60, HSP70, and HSP90. Dorsal rhizotomy, injuring the central axon of the DRG neuron, does not upregulate HSP27 mRNA levels. After peripheral axotomy, HSP27 mRNA and protein are present in small, medium, and large DRG neurons, and HSP27 protein is transported anterogradely, accumulating in the dorsal horn and dorsal columns of the spinal cord, where it persists for several months. Axotomized motor neurons also upregulate HSP27. Only a minority of cultured adult DRG neurons are HSP27-immunoreactive soon after dissociation, but all express HSP27 after 24 hr in culture with prominent label throughout the neuron, including the growth cone. HSP27 differs from most axonal injury-regulated and growth-associated genes, which are typically present at high levels in early development and downregulated on innervation of their targets, in that its mRNA is first detectable in the DRG late in development and only approaches adult levels by postnatal day 21. In non-neuronal cells, HSP27 has been shown to be involved both in actin filament dynamics and in protection against necrotic and apoptotic cell death. Therefore, its upregulation after adult peripheral nerve injury may both promote survival of the injured neurons and contribute to alterations in the cytoskeleton associated with axonal growth.
Leukemia inhibitory factor is an anti-inflammatory and analgesic cytokine.
The mRNA for leukemia inhibitory factor (LIF), a neuroimmune signaling molecule, is elevated during skin inflammation produced by intraplantar injection of complete Freund's adjuvant (CFA). Moreover, although LIF knock-out mice display normal sensitivity to cutaneous mechanical and thermal stimulation compared with wild-type mice, the degree of CFA-induced inflammation in mice lacking LIF is enhanced in spatial extent, amplitude, cellular infiltrate, and interleukin (IL)-1beta and nerve growth factor (NGF) expression. Conversely, local injection of low doses of recombinant LIF diminishes mechanical and thermal hypersensitivity as well as the IL-1beta and NGF expression induced by CFA. These data show that upregulation of LIF during peripheral inflammation serves a key, early anti-inflammatory role and that exogenous LIF can reduce inflammatory hyperalgesia.
Identification of somatostatin sst2(a) receptor expressing neurones in central regions involved in nociception.
Somatostatin is a neuromodulator and neurotransmitter in the central nervous system. Administration of somatostatin to the spinal cord or brain areas involved in nociception has been shown to result in analgesia. Little information is available about the somatostatin receptor types which may be involved in mediating the neuromodulatory and analgesic effects of the peptide. To define the neuronal systems expressing the sst2(a) receptor in brain areas associated with analgesia, immunohistochemical co-localisation studies were carried out in the periaqueductal grey (PAG) and spinal cord using an antibody specific for the sst2(a) receptor. To further define sst2(a) receptor expressing neurones, sst2(a) receptor immunohistochemistry was combined with retrograde tracing using fluorogold. In the PAG, sst2(a) receptor expressing neurones were found to co-express calbindin D28k (36%), the glutamate transporter EAAC-1 (25%), and GABA transporter GAT-1 ( approximately 10%). A total of 65% of sst2(a) positive neurones projected to the thalamus. In the spinal cord, the sst2(a) receptor shows cellular co-localisation with EAAC-1 and GAT-1. Immunohistochemistry and receptor autoradiography using [125I]BIM 23027 after dorsal rhizotomy of the lumbar dorsal roots, L4 and L5, suggests that the somatostatin sst2(a) receptor is not present on primary afferent neurones. Dorsal hemisections of the mid thoracic cord did not alter the immunohistochemical signal for the somatostatin sst2(a) receptor, providing further evidence for an intrinsic localisation of the receptor protein in the dorsal horn of the spinal cord. These data show that the somatostatin sst2(a) receptor exists on morphologically and neurochemically heterogenous neurones and is closely associated with brain areas involved in analgesia and the modulation of nociception.
Deafferentation is insufficient to induce sprouting of A-fibre central terminals in the rat dorsal horn.
The mechanism by which A-fibres sprout into lamina II of the dorsal horn of the adult rat after peripheral nerve injury, a region which normally receives input from noci- and thermoreceptive C-fibres alone, is not known. Recent findings indicating that selective C-fibre injury and subsequent degenerative changes in this region are sufficient to induce sprouting of uninjured A-fibres have raised the possibility that the structural reorganisation of A-fibre terminals is an example of collateral sprouting, in that deafferentation of C-fibre terminals alone in lamina II may be sufficient to cause A-fibre sprouting. Primary afferents of the sciatic nerve have their cell bodies located predominantly in the L4 and L5 dorsal root ganglia (DRGs), and the A-fibres of each DRG have central termination fields that show an extensive rostrocaudal overlap in lamina III in the L4 and L5 spinal segments. In this study, we have found that C-fibres from either DRG have central terminal fields that overlap much less in lamina II than A-fibres in lamina III. We have exploited this differential terminal organisation to produce deafferentation in lamina II of the L5 spinal segment, by an L5 rhizotomy, and then test whether A-fibres of the intact L4 dorsal root ganglion, which terminate within the L5 segment, sprout into the denervated lamina II in the L5 spinal segment. Neither intact nor peripherally injured A-fibres were seen to sprout into denervated lamina II after L5 rhizotomy. Sprouting was only ever seen into regions of lamina II containing the terminals of peripherally injured C-fibres. Therefore, it seems that the creation of synaptic space within lamina II is not the explanation for A-fibre sprouting after peripheral nerve section or crush, emphasising that injury-induced changes in C-fibres and subsequent chemotrophic effects in the superficial dorsal horn are the likely explanation.
Recovery of C-fiber-induced extravasation following peripheral nerve injury in the rat.
Peripheral nerve injury leads to substantial alterations in injured sensory neurons. These include cell death, phenotypic modifications, and regeneration. Primary sensory neurons have recently been shown not to die until a time beyond 4 months following a nerve crush or ligation and this loss is, moreover, limited to cells with unmyelinated axons, the C-fibers. The late loss of C-fibers may be due to a lack of target reinnervation during the regenerative phase. In order to investigate this, we have used a particular peripheral function, unique to C-fibers, as a measure of peripheral reinnervation: an increase in capillary permeability on antidromic activation of C-fibers, i.e., neurogenic extravasation. This was investigated in rats that had received a nerve crush injury 1 to 50 weeks earlier. Some recovery of the capacity of C-fibers to generate extravasation was detected at 8-10 weeks, which increased further at 12-14 weeks, and then plateaued at this level with no further recovery at 30 or 50 weeks. In intact and damaged sciatic nerves, A beta-fibers never induced extravasation. These findings are compatible with the hypothesis that those C-fibers which make it back to their peripheral targets do not subsequently die and those that do not, may die.
Null mutations lacking substance: elucidating pain mechanisms by genetic pharmacology.
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Two sodium channels contribute to the TTX-R sodium current in primary sensory neurons.
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High efficiency gene transfer to the central nervous system of rodents and primates using herpes virus vectors lacking functional ICP27 and ICP34.5.
The safe and efficient use of herpes simplex virus (HSV)-based vectors to deliver genes of potentially therapeutic benefit to the central nervous system will require their effective disablement by the inactivation of viral genes required for lytic growth. Here we report that viruses lacking functional genes for ICP27 (which is required for growth in all cell types) and ICP34.5 (which is required for growth in nondividing cell types) can deliver a marker gene to both the rodent and primate CNS with high efficiency whilst producing relatively minimal damage and having no effect on sodium currents in dorsal root ganglion neurons. Such viruses paradoxically deliver genes at much higher efficiency than the less disabled single mutant lacking ICP34.5 alone and also, as expected, produce less damage in vivo. Moreover, unlike the single mutant lacking ICP27 the double mutant viruses cannot revert to wild-type by acquistion of complimenting gene sequences during growth of virus stocks in vitro on dividing cells expressing ICP27 since artificial expression of ICP34.5 in these cells is not required. Such ICP27-; ICP34.5- viruses thus offer a platform for the development of vectors which are sufficiently safe for ultimate use in human gene therapy.
Growth-associated protein 43 immunoreactivity in the superficial dorsal horn of the rat spinal cord is localized in atrophic C-fiber, and not in sprouted A-fiber, central terminals after peripheral nerve injury.
Peripheral nerve injury induces the up-regulation in dorsal root ganglion cells of growth-associated protein 43 (GAP-43) and its transport to the superficial laminae of the dorsal horn of the spinal cord, where it is located primarily in unmyelinated axons and growth-cone like structures. Peripheral nerve injury also induces the central terminals of axotomized myelinated axons to sprout and form novel synaptic contacts in lamina II of the dorsal horn. To investigate whether the sprouting of A-fiber central terminals into lamina II is the consequence of GAP-43 incorporation into their terminal membranes, we have used an ultrastructural analysis with double labelling to identify the localization of GAP-43 immunoreactivity. Transganglionic transport of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) was used to identify C-fiber terminals. Transganglionic transport of the B fragment of cholera toxin conjugated to horseradish peroxidase (B-HRP) was used to label A-fiber sciatic nerve central terminals in combination with GAP-43 immunocytochemistry. GAP-43 was found to colocalize only with WGA-HRP- and not with B-HRP-labelled synapses or axons. In addition, many single-labelled GAP-43 synapses were observed. Many of the WGA-HRP-labelled terminals that were characterized by degenerative changes were GAP-43 immunoreactive. Our results indicate that peripheral nerve injury induces novel synapse formation of A fibers in lamina II but that up-regulated levels of GAP-43 are present mainly in other axon projections to the superficial dorsal horn.
Intact sciatic myelinated primary afferent terminals collaterally sprout in the adult rat dorsal horn following section of a neighbouring peripheral nerve.
Peripheral nerve section induces sprouting of the central terminals of axotomized myelinated primary afferents outside their normal dorsoventral termination zones in lamina I, III, and IV of the dorsal horn into lamina II, an area that normally only receives unmyelinated C-fiber input. This axotomy-induced regenerative sprouting is confined to the somatotopic boundaries of the injured nerve in the spinal cord. We examined whether intact myelinated sciatic afferents are able to sprout novel terminals into neighbouring areas of the dorsal horn in the adult rat following axotomy of two test nerves, either the posterior cutaneous nerve of the thigh or the saphenous nerve. These peripheral nerves have somatotopically organized terminal areas in the dorsal horn that overlap in some areas and are contiguous in others, with that of the sciatic central terminal field. Two weeks after cutting either the posterior cutaneous or the saphenous nerve, intact sciatic myelinated fibers labelled with the B fragment of cholera toxin conjugated to horseradish peroxidase (B-HRP) sprouted into an area of lamina II normally only innervated by the adjacent injured test nerve. This collateral sprouting was strictly limited, however, to those particular areas of the dorsal horn where the A-fiber terminal field of the control sciatic and the C-fiber terminal field of the injured test nerve overlapped in the dorsoventral plane. No mediolateral sprouting was seen into those areas of neuropil solely innervated by the test nerve. We conclude that intact myelinated primary afferents do have the capacity to collaterally sprout, but that any resultant somatotopic reorganization of central projections is limited to the dorsoventral plane. These changes may contribute to sensory hypersensitivity at the edges of denervated skin.
Tachykinin NK1 receptor antagonist RP67580 attenuates progressive hypersensitivity of flexor reflex during experimental inflammation in rats.
We have now examined whether the tachykinin NK1 receptor is involved in mediating progressive hypersensitivity of spinal flexor motoneurons induced by repeated peripheral stimulation of inflamed tissue in decerebrate-spinal rats. The mechanical threshold of spinal flexor motoneurons was significantly decreased, and the touch- and pinch-evoked responses significantly increased, 48 h after intra-plantar injection of 100 microliters complete Freund's adjuvant. The threshold was further progressively decreased and the touch- and pinch-evoked responses increased over the 80 min testing period. Subcutaneous injection of the tachykinin NK1 receptor antagonist RP67580 (2-[1-imino-2-(2-methoxy phenyl) ethyl]-7,7 diphenyl-4 perhydroisoindolone-(3aR,7aR)) (20 min prior to the beginning of the test) at 1 mg and 10 mg/kg significantly attenuated the progressive decrease of mechanical withdrawal threshold, and the progressive increase of the touch- and pinch-evoked responses. The inactive enantiomer RP68651 (2-[1-imino-2-(2-methoxy phenyl) ethyl]-7,7 diphenyl-4 perhydroisoindolone-(3aS,7aS)) at 1 mg and 10 mg/kg had no significant effect. The present results indicate that substance P and its preferred tachykinin NK1 receptor are involved in mediating progressive hypersensitivity during inflammation.
The progressive tactile hyperalgesia induced by peripheral inflammation is nerve growth factor dependent.
Experimental peripheral inflammation results in cutaneous mechanical hypersensitivity, and repeated low intensity mechanical stimulation of the inflamed skin induces a progressively incrementing hyperalgesia. We have now examined whether the elevation in nerve growth factor (NGF) induced by the inflammation contributes to this progressive hyperalgesia. An i.p. injection of anti-NGF antiserum (5 microliters g-1) 1 h before induction of inflammation by intraplantar complete Freund's adjuvant (CFA) injection and 24 h after, both reduced the basal inflammatory hypersensitivity and significantly attenuated the progressive increase of spontaneous activity, touch-, pinch- and A beta-afferent-evoked responses, and the progressive reduction of the mechanical threshold of biceps femoris/semitendinosus alpha motoneurones normally evoked by repeated (every 5 min) tactile stimulation of the inflamed hindpaw, in decerebrate-spinal rats. NGF contributes, therefore, to the progressive tactile hyperalgesia elicited by repeated touch stimulation of inflamed tissue.