Michael Holmes

Intraspinal and behavioral consequences of nerve growth factor-induced nociceptive sprouting and nerve growth factor-induced hyperalgesia compared in adult rats.

Intraspinal and behavioral events were studied in adult rats with nociceptive nerves that were undergoing collateral sprouting into adjacent denervated skin. This sprouting, which is driven by endogenous nerve growth factor (NGF), did not cause hyperalgesia. For comparison, we studied an exogenous NGF administration that induced hyperalgesia but was too brief to evoke sprouting. When nociceptive nerves sprouted in skin, back‐labeling with wheat germ agglutinin‐conjugated horseradish peroxidase revealed that their projections sprouted in the cord. The sprouted peripheral nerves now activated more c‐Fos‐containing interneurons, which stimulus‐response studies showed was not due to an increased afferent discharge. We attribute the interneuron recruitment to synaptogenesis following the intraspinal sprouting. Nociceptive stimulation of dorsal skin reflexly activates underlying cutaneous trunci muscle (CTM). When a nociceptive field expanded by nerve sprouting, so did the area of the evoked CTM reflex: this implies a recruitment of CTM motoneurons. We interpret this “matching” of response to stimulus as an adaptive phenomenon ensured by an adaptive intraspinal sprouting of the nociceptive projections. Neither the intraspinal changes nor the reflex changes occurred if peripheral sprouting was blocked by systemic anti‐NGF treatment, indicating that the role of endogenous NGF was only in that sprouting. No comparable adaptive events occurred during NGF‐induced hyperalgesia. Neither nociceptive fields nor CTM reflexes were affected; however there was a recruitment of c‐Fos‐expressing interneurons. This recruitment was not explained by peripheral sensitization, and, because sprouting was not involved here, we attribute the recruitment to “synaptic unmasking,” i.e., an increased effectiveness of the preexisting excitatory circuitry. J. Comp. Neurol. 410:73–89, 1999.

ELECTRICAL MUSCLE STIMULATION AFTER IMMEDIATE NERVE REPAIR REDUCES MUSCLE ATROPHY WITHOUT AFFECTING REINNERVATION

Electrical stimulation of denervated muscle has been shown to minimize atrophy and fibrosis and increase force in animal and human models. However, electrical stimulation after nerve repair is controversial due to questions of efficacy.

Spontaneous regeneration of severed optic axons restores mapped visual responses to the adult rat superior colliculus.

To test whether a spontaneous and functional regeneration of severed axons could occur within the adult mammalian central nervous system, a long‐term recovery of microelectrode‐mapped visual response was sought in the superior colliculus (SC) after its total or near‐total abolition by a precise guillotine cut of the retinocollicular pathway. Recoveries were found 3 weeks or later in 15 of the 36 animals studied; in 10 of these recoveries, half or more of the width of the SC was involved. The recovered responses were often activated from within a normally small area of the visual field. Appropriate retinotopic maps were restored. Intraocular horseradish peroxidase tracing revealed a variety of novel optic trajectories, passing around lesions even of totally cut pathways, which eventually terminated in normally retinorecipient layers of those recovered SCs. Such detours could not be explained by a mechanical reorientation of brain structures. When exactly comparable lesions were examined within a few days, there were no detours: severed optic axons faced the cuts. In long‐term animals where responsiveness remained absent, optic axonal reorientations were observed near lesions but the SC was not innervated. Extensive long‐term recoveries were in marked contrast to the occasional rapid ones, found within a few days postlesion, which involved only an outermost silenced border of SC. These were attributed to a rapid reversal of conduction failure in spared, bordering, axons of this topographically organized pathway. The findings support the conclusion that, after they are cut, numbers of optic axons can regenerate to the SC and restore appropriate circuitry therein.

Ceramide Is Responsible for the Failure of Compensatory Nerve Sprouting in Apolipoprotein E Knock-Out Mice

Apolipoprotein E (apoE) is a key transporter of the cholesterol and phospholipids required for membrane synthesis and nerve growth. We now report a virtual absence in apoE knock-out (KO) mice of normal nerve growth factor (NGF)-driven compensatory sprouting of undamaged cutaneous nociceptive nerves. In contrast, NGF-independent regeneration of crushed axons was unaffected. Essentially similar results came from aged wild-type mice. In apoE KO mice, the endogenous sprouting stimulus was suspect, because NGF administration induced normal sprouting; nevertheless, NGF increased normally in denervated skin, transported normally in the axons, and led to phosphorylation of trkA, erk1, and erk2. However, sprouting was restored in apoE KO mice (although not in aged mice) by fumonisin B1, an inhibitor of ceramide synthesis. A shotgun analysis revealed a wide array of changes in individual ceramide species in DRG neurons of apoE KO mice, and the changes for ceramide species OH_N15:0 made it a candidate inhibitor of sprouting (increased in apoE KO mice and normalized by fumonisin B1). Nevertheless, the unknown effects of individual ceramide species on sprouting, as well as the variability of their changed levels in apoE KO mice and how these were affected by fumonisin B1, support a different conclusion. We suggest that absence of apoE expression alters the balance among ceramide species to one that collectively inhibits compensatory sprouting, whereas fumonisin B1 establishes a new balance that allows sprouting. Nontoxic ceramide modulators might usefully promote sprouting and circuitry repair in neurodegenerative disorders in which ceramide species are perturbed, adding to the benefits of reducing ceramide-induced neuronal apoptosis.

Determining the effects of electrical stimulation on functional recovery of denervated rat gastrocnemius muscle using motor unit number estimation

The use of electrical muscle stimulation to treat denervated muscle prior to delayed reinnervation has been widely debated. There is evidence showing both positive and negative results following different protocols of electrical stimulation. In this study we investigated the role electrical stimulation has on muscle reinnervation following immediate and delayed nerve repair using motor unit estimation techniques. Rat gastrocnemius muscle was denervated and repaired using the peroneal nerve either immediately or following three-months with and without electrical stimulation. Motor unit counts, average motor unit sizes, and maximum compound action potentials were measured three-months following per-oneal nerve repair. Motor unit counts in animals that were denervated and stimulated were significantly higher than those that were denervated and not stimulated. Both average motor unit sizes and maximum compound action potentials showed no significant differences between denervated and denervated-stimulated animals. These results provide evidence that electrical stimulation prior to delayed nerve repair increases muscle receptivity to regenerating axons and may be a worthwhile treatment for peripheral nerve injuries.

Sensory nerve cross-anastomosis and electrical muscle stimulation synergistically enhance functional recovery of chronically denervated muscle.

Background: Long-term muscle denervation leads to severe and irreversible atrophy coupled with loss of force and motor function. These factors contribute to poor functional recovery following delayed reinnervation. The authors’ previous work demonstrated that temporarily suturing a sensory nerve to the distal motor stump (called sensory protection) significantly reduces muscle atrophy and improves function following reinnervation. The authors have also shown that 1 month of electrical stimulation of denervated muscle significantly improves function and reduces atrophy. In this study, the authors tested whether a combination of sensory protection and electrical stimulation would enhance functional recovery more than either treatment alone. Methods: Rat gastrocnemius muscles were denervated by cutting the tibial nerve. The peroneal nerve was then sutured to the distal tibial stump following 3 months of treatment (i.e., electrical stimulation, sensory protection, or both). Three months after peroneal repair, functional and histologic measurements were taken. Results: All treatment groups had significantly higher muscle weight (p < 0.05) and twitch force (p < 0.001) compared with the untreated group (denervated), but fiber type composition did not differ between groups. Importantly, muscle weight and force were significantly greater in the combined treatment group (p < 0.05) compared with stimulation or sensory protection alone. The combined treatment also produced motor unit counts significantly greater than sensory protection alone (p < 0.05). Conclusions: The combination treatment synergistically reduces atrophy and improves reinnervation and functional measures following delayed nerve repair, suggesting that these approaches work through different mechanisms. The authors’ research supports the clinical use of both modalities together following peripheral nerve injury.

Neotrofin, a novel purine that induces NGF-dependent nociceptive nerve sprouting but not hyperalgesia in adult rat skin

We report peripheral actions in rats of Neotrofin, a purine derivative of therapeutic interest. Systemic injections mimicked NGF in eliciting sprouting of nociceptive nerves without affecting their regeneration. The sprouting was prevented by anti-NGF treatment, implicating endogenous NGF. We detected no Neotrofin-induced increases in cutaneous NGF levels or in retrograde NGF transport. In contrast, both NGF and phosphorylation of trkA increased significantly in DRGs, with a marginal appearance of phosphorylated trkA in axons. We conclude that the DRG effects of Neotrofin are responsible for its induction of sprouting. Neotrofin also induced a striking phosphorylation of axonal erk 1 and 2, which was, however, unaffected by anti-NGF treatment. We suggest that this NGF-independent MAP kinase activation is involved in nonsprouting functions of Neotrofin such as neuroprotection. Unlike injected NGF, Neotrofin did not induce hyperalgesia, supporting its candidacy as a treatment for peripheral neuropathies like those induced by diabetes and anticancer chemotherapy.