Functional motoneurons develop from human neural stem cell transplants in adult rats.

We have shown previously that primed human fetal neural stem cells, after transplantation into rat spinal cords, differentiated into cholinergic motoneurons that sent axons to contact medial gastrocnemius myocytes. Here we demonstrate that (i) axons from the transplanted cells are cholinergic and myelinated, (ii) putative synapses form on transplanted somata and dendrites in the ventral horn, (iii) human fetal neural stem cells transplantation led to normal electromyograms from medial gastrocnemius muscles, and (iv) the gait of transplanted animals was much improved. Accumulatively, our data indicate that some transplanted human fetal neural stem cells in adult motoneuron-deficient ventral horns differentiate into relatively normal motoneurons that are integrated into spinal and peripheral circuitry. These findings are steps towards the long-term goal of providing stem cell transplants for motoneuron loss.

Fos, nociception and the dorsal horn.

The protooncogene c-fos is rapidly activated after noxious stimuli to express the protein Fos in spinal dorsal horn neurons that are in the 'correct' locations for nociceptive information transfer. As such, therefore, mapping Fos expression in these neurons is at present the best global marker for efficiently locating populations of neurons in the awake animal that respond to nociceptive input. This allows, among other things, precise behavioral measurements to be correlated with Fos expression. Two arenas where mapping dorsal horn Fos expression has made a major impact are in the anatomy of nociceptive systems and as a useful assay for the analgesic properties of various therapeutic regimens. Also Fos expression is the only way to map populations of neurons that are responding to non-localized input such as withdrawal after addiction and vascular occlusion. Another insight is that it shows a clear activation of neurons in superficial 'pain-processing' laminae by innocuous stimuli after nerve lesions, a finding that presumably bears on the allodynia that often accompanies these lesions. It is to be understood, however, that the Fos localizations are not sufficient unto themselves, but the major function of these studies is to efficiently locate populations of cells in nociceptive pathways so that powerful anatomic and physiologic techniques can be brought to bear efficiently. Thus, the purpose of this review is to summarize the studies whose numbers are geometrically expanding that deal with Fos in the dorsal horn and the conclusions therefrom.

Blocking caspase activity prevents transsynaptic neuronal apoptosis and the loss of inhibition in lamina II of the dorsal horn after peripheral nerve injury.

We show that transsynaptic apoptosis is induced in the superficial dorsal horn (laminas I-III) of the spinal cord by three distinct partial peripheral nerve lesions: spared nerve injury, chronic constriction, and spinal nerve ligation. Ongoing activity in primary afferents of the injured nerve and glutamatergic transmission cause a caspase-dependent degeneration of dorsal horn neurons that is slow in onset and persists for several weeks. Four weeks after spared nerve injury, the cumulative loss of dorsal horn neurons, determined by stereological analysis, is >20%. GABAergic inhibitory interneurons are among the neurons lost, and a marked decrease in inhibitory postsynaptic currents of lamina II neurons coincides with the induction of apoptosis. Blocking apoptosis with the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD) prevents the loss of GABAergic interneurons and the reduction of inhibitory currents. Partial peripheral nerve injury results in pain-like behavioral changes characterized by hypersensitivity to tactile or cold stimuli. Treatment with zVAD, which has no intrinsic analgesic properties, attenuates this neuropathic pain-like syndrome. Preventing nerve injury-induced apoptosis of dorsal horn neurons by blocking caspase activity maintains inhibitory transmission in lamina II and reduces pain hypersensitivity.

Reactive oxygen species (ROS) play an important role in a rat model of neuropathic pain.

Reactive oxygen species (ROS) are free radicals produced in biological systems that are involved in various degenerative brain diseases. The present study tests the hypothesis that ROS also play an important role in neuropathic pain. In the rat spinal nerve ligation (SNL) model of neuropathic pain, mechanical allodynia develops fully 3 days after nerve ligation and persists for many weeks. Systemic injection of a ROS scavenger, phenyl-N-tert-butylnitrone (PBN), relieves SNL-induced mechanical allodynia in a dose-dependent manner. Repeated injections cause no development of tolerance or no loss of potency. Preemptive treatment with PBN is also effective in preventing full development of neuropathic pain behavior. Systemic injection was mimicked by intrathecal injection with a little less efficacy, while intracerebroventricular administration produced a much smaller effect. These data suggest that PBN exerts its anti-allodynic action mainly by spinal mechanisms. Systemic treatment with other spin-trap reagents, 5,5-dimethylpyrroline-N-oxide and nitrosobenzene, showed similar analgesic effects, suggesting that ROS are critically involved in the development and maintenance of neuropathic pain. Thus this study suggests that systemic administration of non-toxic doses of free radical scavengers could be useful for treatment of neuropathic pain.

Somatostatin modulates the transient receptor potential vanilloid 1 (TRPV1) ion channel.

Activation of peripheral somatostatin receptors (SSTRs) inhibits sensitization of nociceptors, thus having a short term or phasic effect [Pain 90 (2001) 233] as well as maintaining a tonic inhibitory control over nociceptors [J Neurosci 21 (2001) 4042]. The present study provides several lines of evidence that an important mechanism underlying SSTR modulation of nociceptors is regulation of the transient receptor potential vanilloid 1 ion channel (TRPV1, formerly the VR1 receptor). Double labeling of L5 dorsal root ganglion cells demonstrates that approximately 60% of SSTR2a-labeled cells are positive for TRPV1. Conversely, approximately 33% of TRPV1-labeled cells are positive for SSTR2a. In vivo behavioral studies demonstrate that intraplantar injection of 20.0 but not 2.0 microM octreotide (OCT, SSTR agonist) significantly reduces capsaicin (CAP, a ligand for TRPV1) -induced flinching and lifting/licking behaviors. This occurs through local activation of SSTRs in the injected hindpaw and is reversed following co-application of the SSTR antagonist cyclo-somatostatin (c-SOM). In vitro studies using a skin-nerve preparation demonstrate that activation of peripheral SSTRs on nociceptors with 20.0 microM OCT significantly reduces CAP-induced activity and can prevent CAP-induced desensitization. Furthermore, blockade of peripheral SSTRs with c-SOM dramatically enhances CAP-induced behaviors and nociceptor activity, demonstrating SSTR-induced tonic inhibitory modulation of TRPV1. Finally, TRPV1 does not appear to be under tonic opioid receptor control since the opioid antagonist naloxone does not change CAP-induced excitation and does not effect OCT-induced inhibition of CAP responses. These data strongly suggest that SSTRs modulate nociceptors through phasic and tonic regulation of peripheral TRPV1 receptors.

Differential expression of tetrodotoxin-resistant sodium channels Nav1.8 and Nav1.9 in normal and inflamed rats.

In an attempt to understand mechanisms underlying peripheral sensitization of primary afferent fibers, we investigated the presence of the tetrodotoxin-resistant Na+ channel subunits Nav1.8 (SNS) and Nav1.9 (SNS2) on axons in digital nerves of normal and inflamed rat hindpaws. In normal animals, 14.3% of the unmyelinated and 10.7% of the myelinated axons labeled for the Nav1.8 subunit. These percentages significantly increased in 48 h inflamed animals to 22.0% (1.5-fold increase) and 57.5% (6-fold increase) for unmyelinated and myelinated axons, respectively. In normal animals, Nav1.9 labeled 9.9% of the unmyelinated and 2.1% of the myelinated axons and following inflammation, the proportion of Nav1.9-labeled unmyelinated axons significantly decreased to 3.0% with no change in the proportion of labeled myelinated axons. These data indicate that Nav1.8 and Nav1.9 subunits are transported to the periphery in normal animals and are differentially regulated during inflammation. The massive increase in Nav1.8 expression in myelinated axons suggests that these may contribute to peripheral sensitization and inflammatory hyperalgesia.

Control of postganglionic sympathetic efferent fibers by neurokinin 1 receptors in rats.

Substance P (SP) is released peripherally from nociceptive terminals and has a direct effect on vascular cells. The present study suggests an indirect effect as well. In normal rats and rats with one hindpaw inflamed for 48 h following intraplantar injection of complete Freund's adjuvant, neurokinin 1 (NK1) receptors were immunohistochemically localized on postganglionic sympathetic axons. The percentage of NK1-labeled axons in the gray rami from normal rats was 35.0+/-5.0; in inflamed rats this percentage increased significantly to 49.1+/-2.3. These data suggest a sensory-sympathetic reflex, where SP activates these peripheral sympathetic receptors with subsequent release of noradrenaline and other compounds to affect vascular cells indirectly. This control is enhanced after inflammation.

Disruption of ErbB receptor signaling in adult non-myelinating Schwann cells causes progressive sensory loss.

Here we studied the role of signaling through ErbB-family receptors in interactions between unmyelinated axons and non-myelinating Schwann cells in adult nerves. We generated transgenic mice that postnatally express a dominant-negative ErbB receptor in non-myelinating but not in myelinating Schwann cells. These mutant mice present a progressive peripheral neuropathy characterized by extensive Schwann cell proliferation and death, loss of unmyelinated axons and marked heat and cold pain insensitivity. At later stages, C-fiber sensory neurons die by apoptosis, a process that may result from reduced GDNF (glial cell line-derived neurotrophic factor) expression in the sciatic nerve. Neuregulin 1 (NRG1)-ErbB signaling mediates, therefore, reciprocal interactions between non-myelinating Schwann cells and unmyelinated sensory neuron axons that are critical for Schwann cell and C-fiber sensory neuron survival. This study provides new insights into ErbB signaling in adult Schwann cells, the contribution of non-myelinating Schwann cells in maintaining trophic support of sensory neurons, and the possible role of disrupted ErbB signaling in peripheral sensory neuropathies.

Region-specific generation of cholinergic neurons from fetal human neural stem cells grafted in adult rat.

Pluripotent or multipotent stem cells isolated from human embryos or adult central nervous system (CNS) may provide new neurons to ameliorate neural disorders. A major obstacle, however, is that the majority of such cells do not differentiate into neurons when grafted into non-neurogenic areas of the adult CNS. Here we report a new in vitro priming procedure that generates a nearly pure population of neurons from fetal human neural stem cells (hNSCs) transplanted into adult rat CNS. Furthermore, the grafted cells differentiated by acquiring a cholinergic phenotype in a region-specific manner. This technology may advance stem cell-based therapy to replace lost neurons in neural injury or neurodegenerative disorders.

Inflammation-induced up-regulation of neurokinin 1 receptors in rat glabrous skin.

Peripheral inflammation sensitizes primary afferent fibers causing lowered thresholds and increased responses to noxious input. One mechanism for sensitization might be increased expression of receptors whose activation results in nociceptor activity. Accordingly, the present study demonstrates that 15.6% of unmyelinated primary afferent axons in rat glabrous skin express the neurokinin 1 (NK1) receptor. At 48 h following hindpaw inflammation with complete Freund's adjuvant, the proportion of unmyelinated axons expressing NK1 receptors significantly increases to 23.6%. This implies a considerable upregulation of NK1 receptor synthesis in the dorsal root ganglia with subsequent transport to peripheral nociceptive terminals. Antagonizing peripheral NK1 receptors locally would be effective in reducing inflammatory pain by reducing neural transduction in NK1-expressing nociceptors as well as lessening the inflammatory vascular effects of peripheral substance P.

Somatostatin receptors on peripheral primary afferent terminals: inhibition of sensitized nociceptors.

Somatostatin (SST) is in primary afferent neurons and reduces vascular and nociceptive components of inflammation. SST receptor (SSTR) agonists provide analgesia following intrathecal or epidural administration in humans, but neurotoxicity in the central nervous system (CNS) has been reported in experimental animals. With the rationale that targeting peripheral SSTRs would provide effective analgesia while avoiding CNS side effects, the goals of the present study are to investigate the presence of SSTRs on peripheral primary afferent fibers and determine the behavioral and physiological effects of the SST agonist octreotide (OCT) on formalin-induced nociception and bradykinin-induced primary afferent excitation and sensitization in the rat. The results demonstrate that: (1) SSTR2as are present on 11% of peripheral primary afferent sensory fibers in rat glabrous skin; (2) intraplantar injection of OCT reduces formalin-induced nociceptive behaviors; (3) OCT reduces, in a dose-dependent fashion, responses to thermal stimulation in C-mechanoheat sensitive fibers; and (4) OCT reduces the responses of C-mechanoheat fibers to bradykinin-induced excitation and sensitization to heat. Each of these actions can be reversed following co-injection of OCT with the SSTR antagonist cyclo-somatostatin (c-SOM). Thus, activation of peripheral SSTRs reduces both inflammatory pain and the activity of sensitized nociceptors, avoids deleterious CNS side effects and may be clinically useful in the treatment of pain of peripheral origin.

Assaying structural changes after nerve damage, an essay on quantitative morphology.

Morphologic changes, such as cell loss or sprouting after nerve lesions, presumably have an important bearing on chronic pain states. Accordingly, unbiased estimates of such things as cell or synapse numbers are necessary to evaluate these difficulties. Basic principles for obtaining such estimates are in dispute, however, so the present paper is an essay on these techniques with the aim of making it easier for nondevotees of these methods to make accurate assessments when such data are reported. The basic points are that, with the exception of serial reconstructions, which are very inefficient, older techniques to determine cell and synapse numbers from histological sections make geometric assumptions that are almost never verified or quantified. Accordingly, the geometrical biases that arise from these assumptions are not assessed. Fortunately, recent stereological techniques do not depend on these assumptions and so are free from the above biases. As such, these represent a technological advance. It must be remembered, however, that all methods are subject to potential observational, methodological and sampling biases.

Adrenergic sensitivity of the sensory receptors modulating mechanical allodynia in a rat neuropathic pain model.

This study focuses on changes in adrenergic sensitivity in untransected sensory axons that innervate an area of skin made neuropathic by transection of neighboring nerves. The segmental nerve injury model is favorable for this since all axons in the L5 and L6 nerves are transected whereas the L4 axons are intact. Earlier findings are that pain behaviors develop after this injury and that these behaviors are ameliorated by sympathectomy. The present study shows that behavior indicating mechanical allodynia can be rekindled after sympathectomy by intradermal norepinephrine and alpha-2 but not alpha-1 adrenergic ligands and the rekindling can be blocked by alpha-2 but not alpha-1 adrenergic antagonists. By contrast neither intradermal norepinephrine nor other adrenergic agonists or antagonists have any demonstrable effects in the normal or after either neuropathic surgery or sympathectomy alone. These data suggest that the combination of neuropathic surgery and sympathectomy results in an upregulation of active alpha-2 adrenergic receptors on the undamaged sensory axons that provide the remaining sensory innervation to a neuropathic area partially denervated by segmental nerve lesions. These changes on undamaged axons presumably compliment similar changes on the transected axons and, thus play a role in the development of neuropathic pain.

Is large myelinated fiber loss associated with hyperalgesia in a model of experimental peripheral neuropathy in the rat?

Recently it has been shown that placement of 4 loose chromic gut sutures around the rat sciatic nerve produces hyperalgesia. A possible mechanism underlying this hyperalgesia is a preferential loss of large myelinated fibers. A difficulty, however, is that neuropathic symptoms are not static and the time course of the axon loss has not been determined. To remedy this deficit, the present study relates axonal changes to the behavior of the animal at various times after induction of the neuropathy. The findings are that a loss of all axon types, with a preferential loss of large myelinated axons, is associated with the development of heat hyperalgesia. As the axon loss progresses, however, the hyperalgesia lessens. In addition, at 28 days post surgery there are essentially no large myelinated axons in the distal segment, but the signs of hyperalgesia have almost resolved. These findings indicate that the onset of the hyperalgesia is accompanied by a preferential loss of large fibers and by a lesser but still substantial loss of small myelinated and unmyelinated axons. The subsequent course of the hyperalgesia, however, is not in any obvious way related to the proportions of large myelinated fibers in the affected nerve.

Further evidence for the existence of long ascending unmyelinated primary afferent fibers within the dorsal funiculus: effects of capsaicin.

The present study provides further evidence in support of the hypothesis that there is a fine primary afferent system in the dorsal funiculi by determining the effects of capsaicin (8-methyl-N-vanillyl-6-noneamide) on unmyelinated fibers in the cervical fasciculus gracilis of the rat. The neurolytic effect of this procedure was demonstrated by showing an 89% decrease in the number of unmyelinated fibers in the S2 dorsal roots of the experimental animals. Consequently, we feel that unmyelinated primary afferent fibers are largely removed from these animals. Neonatal administration of capsaicin (50 mg/kg) caused a 54% decrease in the number of unmyelinated fibers in the C3 fasciculus gracilis but no significant change in myelinated fiber numbers. The data provide further evidence for the existence of a significant primary afferent unmyelinated fiber system in the dorsal funiculus and suggest a role for the dorsal funiculi in the transmission of noxious information.