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.

The peripheral branch of primary sensory neurons regenerates after injury, but there is no regeneration when their central branch is severed by spinal cord injury. Here we show that microinjection of a membrane-permeable analog of cAMP in lumbar dorsal root ganglia markedly increases the regeneration of injured central sensory branches. The injured axons regrow into the spinal cord lesion, often traversing the injury site. This result mimics the effect of a conditioning peripheral nerve lesion. We also demonstrate that sensory neurons exposed to cAMP in vivo, when subsequently cultured in vitro, show enhanced growth of neurites and an ability to overcome inhibition by CNS myelin. Thus, stimulating cAMP signaling increases the intrinsic growth capacity of injured sensory axons. This approach may be useful in promoting regeneration after spinal cord injury.

Pain is normally evoked only by stimuli that are sufficiently intense to activate high-threshold A(delta) and C sensory fibres, which relay the signal to the spinal cord. Peripheral inflammation leads to profoundly increased pain sensitivity: noxious stimuli generate a greater response and stimuli that are normally innocuous elicit pain. Inflammation increases the sensitivity of the peripheral terminals of A(delta) and C fibres at the site of inflammation. It also increases the excitability of spinal cord neurons, which now amplify all sensory inputs including the normally innocuous tactile stimuli that are conveyed by low-threshold A(beta) fibres. This central sensitization has been attributed to the enhanced activity of C fibres, which increase the excitability of their postsynaptic targets by releasing glutamate and the neuropeptide substance P. Here we show that inflammation results in A(beta) fibres also acquiring the capacity to increase the excitability of spinal cord neurons. This is due to a phenotypic switch in a subpopulation of these fibres so that they, like C-fibres, now express substance P. A(beta) fibres thus appear to contribute to inflammatory hypersensitivity by switching their phenotype to one resembling pain fibres, thereby enhancing synaptic transmission in the spinal cord and exaggerating the central response to innocuous stimuli.

Protein kinase C ␥ (PKC␥), which is concentrated in interneurons of the inner part of lamina II of the dorsal horn, has been implicated in injury-induced allodynia, a condition wherein pain is produced by innocuous stimuli. Although it is generally assumed that these interneurons receive input from the nonpeptidergic, IB4-positive subset of nociceptors, the fact that PKC␥ cells do not express Fos in response to noxious stimulation suggests otherwise. Here, we demonstrate that the terminal field of the nonpeptidergic population of nociceptors, in fact, lies dorsal to that of PKC␥ interneurons. There was also no overlap between the PKC␥-expressing interneurons and the transganglionic tracer wheat germ agglutinin which, after sciatic nerve injection, labels all unmyelinated nociceptors. However, transganglionic transport of the ␤-subunit of cholera toxin, which marks the medium-diameter and large-diameter myelinated afferents that transmit non-noxious information, revealed extensive overlap with the layer of PKC␥ interneurons. Furthermore, expression of a transneuronal tracer in myelinated afferents resulted in labeling of PKC␥ interneurons. Light and electron microscopic double labeling further showed that the VGLUT1 subtype of vesicular glutamate transmitter, which is expressed in myelinated afferents, marks synapses that are presynaptic to the PKC␥ interneurons. Finally, we demonstrate that a continuous non-noxious input, generated by walking on a rotarod, induces Fos in the PKC␥ interneurons. These results establish that PKC␥ interneurons are activated by myelinated afferents that respond to innocuous stimuli, which suggests that injury-induced mechanical allodynia is transmitted through a circuit that involves PKC␥ interneurons and non-nociceptive, VGLUT1-expressing myelinated primary afferents.

The peripheral axonal branch of primary sensory neurons readily regenerates after peripheral nerve injury, but the central branch, which courses in the dorsal columns of the spinal cord, does not. However, if a peripheral nerve is transected before a spinal cord injury, sensory neurons that course in the dorsal columns will regenerate, presumably because their intrinsic growth capacity is enhanced by the priming peripheral nerve lesion. As the effective priming lesion is made before the spinal cord injury it would clearly have no clinical utility, and unfortunately, a priming lesion made after a spinal cord injury results in an abortive regenerative response. Here, we show that two priming lesions, one made at the time of a spinal cord injury and a second 1 week after a spinal cord injury, in fact, promote dramatic regeneration, within and beyond the lesion. The first lesion, we hypothesize, enhances intrinsic growth capacity, and the second one sustains it, providing a paradigm for promoting CNS regeneration after injury.primary afferents ͉ dorsal columns ͉ neurite outgrowth ͉ sprouting ͉ priming T here is little evidence for significant axonal regeneration after spinal cord injury in the adult (1). This failure to regenerate has been attributed to both intrinsic factors, such as the low inherent capacity of adult neurons to grow, and extrinsic factors in the environment of the lesion. The latter include myelin-associated molecules, such as NOGO (2, 3), myelinassociated glycoprotein (4, 5), oligodendrocyte myelin glycoprotein (6, 7), and chondroitin sulfate proteoglycan (8, 9), which inhibit growth, and an astrocyte-based scar that gradually develops at the lesion site (10, 11).We previously showed that it is possible to induce growth of damaged dorsal column (DC) axons beyond (i.e., rostral to) a spinal cord lesion, but only when the manipulation that enhances growth (namely a priming͞conditioning lesion of the sciatic nerve) is made before the spinal cord injury. When we made the sciatic nerve transection concurrently with the DC lesion, there was growth, but only into the spinal cord lesion site, never beyond it (12). Microinjection of a cAMP analogue also enhanced growth, provided it was injected before the DC lesion (13,14). If the sciatic cut was performed 1 or 2 weeks after the spinal cord injury (postpriming) there was absolutely no growth. The tips of the sensory afferents formed large endbulbs that never penetrated the lesion site (12). Taken together, these results demonstrated that the intrinsic growth capacity of the neurons, which we hypothesize can be enhanced by a sciatic nerve lesion, is an important factor that contributes to longdistance regeneration in the spinal cord. The timing of a priming͞conditioning lesion is critical.The regeneration that we observed in those studies was significant, but clearly it would have no clinical utility. For manipulations that enhance regeneration to have clinical utility, they must be effective when performed at the time of or after the spinal cord injury. T...

whose main function is to convey information from and Harvard Medical School the periphery to the central nervous system. This infor-Charlestown Navy Yard mation is carried by two types of fibers; unmyelinated Charlestown, Massachusetts 02129 C-fibers, which are nociceptors and thermoreceptors, and myelinated A-fibers, which are a combination of Elucidating the function of particular neurotransmitters low threshold mechanoreceptors and high threshold and their receptors has traditionally relied upon pharmamechano-and thermoreceptors. Although modalitycological approaches using compounds that act as spespecific sensory neurons exist, no modality-specific cific agonists or antagonists at defined receptors on the chemical phenotype has been discovered. Substance pre-and postsynaptic membrane. A recent tool has been P immunoreactivity is found in ‫%02ف‬ of lumbar dorsal added to the armamentarium for studying synaptic funcroot ganglia (DRG) neurons, almost all of which are small tion: genetic manipulations, particularly null mutations neurons that also express the nerve growth factor (NGF) or "knockouts" that delete specific ligands or their rereceptor TrkA. Substance P is shipped from the cell ceptors. It is appropriate, though, to ask what the genetic bodies of DRG neurons into their peripheral and central approach adds to the information that can be gleaned by terminals. We will first discuss the actions of substance classic synaptic pharmacology, and to consider whether P in the periphery, and then centrally, in the spinal cord. "genetic pharmacology" can unambiguously define the Substance P in Neurogenic Inflammation overall function of an individual transmitter/neuromodu-When peripheral terminals are depolarized by approlator in complex neurobiological systems. The neuropriate mechanical, thermal, or chemical stimuli, subpeptide substance P and its receptor NK1 are worth stance P, along with calcitonin gene-related peptide examining in this context, since mice with targeted mu-(CGRP), is released and acts on postcapillary venules tations deleting the preprotachykinin gene that proto produce increased permeability (substance P) and duces substance P and neurokinin A (Zimmer et al., dilatation (CGRP), a phenomenon known as neurogenic 1998; Cao et al., 1998) and the substance P receptor NK1 inflammation. The increased permeability produced by (De Felipe et al., 1998) have recently been generated.substance P is associated with an extravasation of In 1977, Tom Jessell and Les Iversen proposed an plasma proteins from the intravascular to the extracelluintriguing model for synaptic transmission from high lar compartment and is uniformly reduced by NK1 recepthreshold nociceptive primary sensory neurons (C-fibers) tor antagonists. The functional significance of neuroto second-order neurons in the spinal cord, where the genic extravasation has been hotly debated; a role for release of substance P, acting as a primary afferent it has been suggested in migraine, asthma, gastrointestitransmitter, was regulated by opioids. D...

one whose complexity cannot be minimized, and which Department of Anesthesia needs to be confronted with a realistic sense not only Massachusetts General Hospital and of what is needed but also what is possible. Clinical Harvard Medical School intervention clearly will be contingent on our under-Charlestown, Massachusetts 02129 standing the neurobiology of regeneration. Regeneration Failure Successful regeneration depends upon the ability of Humpty Dumpty sat on a wall, Humpty Dumpty had a injured axons to survive, regrow, and reconnect with great fall, all the King's horses and all the King's men their original targets, processes integral to normal develcouldn't put Humpty together again. opment. Why then is the regenerative response abortive Spinal Cord Injury in the adult mammalian CNS? There are three explana-In view of the anatomical arrangement of its ascending tions: death of the injured neurons, an inability of differand descending fiber tracts, even local limited injury to entiated adult neurons to initiate or maintain axonal the spinal cord typically has devastating consequences.growth, and a lack of an environment permissive for Communication between that part of the body below such growth (Figure 1). the level of injury and the brain is disturbed, resultingThe inability of the CNS environment to support in permanent para-or quadriplegia and an equivalent growth appears to be due to both the presence of inhibiloss of sensation. These motor and sensory deficits are tory and a lack of growth-promoting signals. There are usually accompanied by positive symptoms, including three sources of such signals; on cells, in the extracellupain and exaggerated motor and autonomic reflexes. lar matrix, and diffusible molecules. One major source Treatment for traumatic spinal cord injury has until of inhibition is myelin. Schwab and colleagues demonnow only had two aims, both conservative. The first at strated some years ago that a monoclonal antibody genthe time of injury is to prevent further damage by higherated against central myelin, IN-1, improved neuronal dose steroids and, where appropriate, stabilizing the regeneration (reviewed by Tatagiba et al., 1997). Since vertebral column and removing compressive tumors and then, myelin-associated glycoprotein (MAG) has been any source of infection. The second is rehabilitative, shown to be a myelin-associated growth-inhibitory molecule (reviewed by Fawcett and Asher, 1999). Recently, teaching the patient and their family to live with this a major achievement has been the cloning and expresdisability by optimizing intact function and reducing sion of a novel protein, Nogo-A, that is the specific target problems like poor ventilation, bedsores, bladder infecfor IN-1's action (Chen et al., 2000; GrandPre et al., 2000; tions, joint fixation, and muscle wastage. Prinjha et al., 2000). Only a small portion of Nogo-A is What are the realistic prospects for a much more amexpressed on the surface (GrandPre et al., 2000), and bitious treatment objective aimed at promoting regene...