Carl Molander

The growth-associated protein GAP-43 appears in dorsal root ganglion cells and in the dorsal horn of the rat spinal cord following peripheral nerve injury.

When adult dorsal root ganglion cells are dissociated and maintained in vitro, both the small dark and the large light neurons show increases in the growth-associated protein GAP-43, a membrane phosphoprotein associated with neuronal development and plasticity. Immunoreactivity for GAP-43 appears in the cytoplasm of the cell bodies as early as 3.5 h post axotomy and is present in neurites and growth cones as soon as they develop. At early stages of culture (4 h to eight days) satellite/Schwann cells are also immunoreactive for GAP-43. Neurons in isolated whole dorsal root ganglion maintained in vitro become GAP-43-immunoreactive between 2 and 3 h after axotomy. It takes three days however, after cutting or crushing the sciatic nerve in adult rats in vivo, for GAP-43 immunoreactivity to appear in the axotomized dorsal root ganglion cells. GAP-43 immunoreactivity can be detected in the central terminals of primary afferent neurons in the superficial laminae of the dorsal horn of the lumbar enlargement four days after sciatic cut or crush. The intensity of the GAP-43 staining reaches a peak at 21 days and becomes undetectable nine weeks following crush injury and 36 weeks following sciatic nerve cut. The pattern of GAP-43 staining is identical to the distribution of sciatic small-calibre afferent terminals. Little or no staining is present in the deep dorsal horn, but GAP-43 does appear in the ipsilateral gracile nucleus 22 days after sciatic injury. In investigating the mechanism of GAP-43 regulation, blockade of axon transport in the sciatic nerve with vinblastine (10(-5) M-10(-4) M) or capsaicin (1.5%) was found to produce a pattern of GAP-43 immunoreactivity in the dorsal horn identical to that found with crush, while electrical stimulation of the sciatic nerve had no effect. Axotomy of primary sensory neurons or the interruption of axon transport in the periphery therefore acts to trigger GAP-43 production in the cell body. The GAP-43 is transported to both the peripheral and the central terminals of the afferents. In the CNS the elevated GAP-43 levels may contribute to an inappropriate synaptic reorganization of afferent terminals that could play a role in the sensory disorders that follow nerve injury.

Substance P-, somatostatin- and calcitonin gene-related peptide-like immunoreactivity and fluoride resistant acid phosphatase-activity in relation to retrogradely labeled cutaneous, muscular and visceral primary sensory neurons in the rat

The distribution of several peptides in cutaneous, muscular and visceral primary sensory neurons was investigated in the adult rat. The fluorescent dye Fast blue was applied to the proximal ends of transected saphenous (cutaneous), gastrocnemius (muscular) and greater splanchnic (visceral) nerves. Sections from corresponding dorsal root ganglia were incubated for simultaneous indirect immunocytochemical demonstration of calcitonin gene-related peptide (CGRP)-, substance P (SP)- or somatostatin (SOM)-like immunoreactivity (-LI) and Fast blue. In addition, the presence of fluoride resistant acid phosphatase (FRAP)-enzyme activity (-EA) in retrogradely Fast blue-labeled saphenous and gastrocnemius nerves was investigated by subsequent enzyme cytochemical analysis. The results revealed the presence of CGRP-LI, SP-LI, SOM-LI and FRAP-EA in cell bodies of primary sensory neurons which project to the saphenous and gastrocnemius nerves. CGRP-LI and SP-LI, but not SOM-LI, were found in splanchnic sensory neurons. The vast majority of the visceral sensory neurons were found to contain CGRP-LI.

Prolonged relief of neuralgia after regional anesthetic blocks. A call for further experimental and systematic clinical studies.

&NA; Thirty‐eight consecutive patients with neuralgia after peripheral nerve injury were treated with one or two series of peripheral local anesthetic blocks. All patients experienced an initial total relief of ongoing pain for 4–12 h. Evoked pain (hyperalgesia or allodynia), which occurred in 17 patients, was blocked simultaneously with the spontaneous pain. In 18 patients the analgesia outlasted the conduction block and there was a period of complete pain relief of 12–48 h in 13 patients and of 2–6 days in the other 5. In 8 patients there was a second phase of analgesia of 4 h to 6 days duration occurring within 12 h of pain recurrence. Thus, mono‐ or biphasic prolonged complete analgesia occurred in 25 out of 38 patients. A prolonged analgesia may be the result of a central action of the local anesthetic at the spinal level after intra‐axonal incorporation and centripetal axoplasmic transport. To test this hypothesis, an experimental study with [3H]lidocaine was performed in 6 rats. The radioactive local anesthetic was injected into one hind limb foot with the other side serving as a control. Tissue samples from the peripheral nerve, nerve root and the lumbosacral spinal cord segment were analyzed for radioactivity using a scintillation counter technique at various time intervals after the [3H]lidocaine injection. There was a low grade of activity in all samples and no difference between the test side and the control side. Thus these experiments provided no evidence in support of this hypothesis. Various alternative peripheral and central mechanisms are discussed. Further studies specifically directed to these alternatives and with longitudinal controls are prompted.

Changing pattern of c-fos expression in spinal cord neurons after electrical stimulation of the chronically injured sciatic nerve in the rat

Immunocytochemical technique was used to study the distribution of c-FOS protein immunoreactive cells in the spinal cord and gracile nuclei 2 h after electrical stimulation of the sciatic nerve in ketamine/xylazine/acepromazine-anesthetized adult rats. Quantitative examination of the c-fos-labeled cells in the spinal cord laminae was made in unoperated and sham operated controls, after sciatic nerve transection without electrical stimulation, and after electrical stimulation at C-fiber or A alpha/beta-fiber intensity, both in normal animals and at various survival times after chronic sciatic nerve injury (transection and ligation) or crush. Unoperated animals showed very few c-fos-labeled cells, and sham operated controls showed labeled cells located mainly outside the sciatic nerve projection territory. A small increase in number of c-fos protein positive cells was seen after sciatic nerve transection without electrical stimulation. Stimulation of the normal sciatic nerve at C-fiber intensity resulted in c-fos protein-positive cells within the sciatic projection territory in the ipsilateral dorsal horn. Labeled cells were seen in all spinal cord laminae except lamina IX, with the vast majority in lamina I and outer lamina II. No labeled cells were seen in the gracile nucleus. Stimulation at A alpha/beta fiber intensity resulted in no or only a very small number of c-fos-positive neurons. Electrical stimulation of the injured sciatic nerve at C-fiber intensity, using the uninjured contralateral side as control, resulted in significant decreases in c-fos-immunoreactive cells in lamina I plus the outer portion of lamina II at 12 and 39 days survival after injury. A non-significant decrease was seen in these laminae also after 21 days. Significant increases were seen in laminae III and IV at 21 days. Decreases in laminae V, VI and more ventral laminae were significant at 21 and 39 days after injury. At longer survival times, the difference between the normal and injured side seen weeks after injury tended to disappear. Stimulation at A alpha/beta fiber intensity 21 days after injury resulted in increases in the numbers of labeled cells in ipsilateral laminae II, III and IV and in the gracile nucleus. Sciatic nerve stimulation after crush injury resulted in more variable side differences, with tendencies for the same alterations as those noted after chronic transection-ligation.(ABSTRACT TRUNCATED AT 400 WORDS)

A quantitative analysis of the microglial cell reaction in central primary sensory projection territories following peripheral nerve injury in the adult rat.

The time course of the microglial cell reaction in central nervous system primary sensory projection territories has been examined following peripheral nerve injury in the adult rat using qualitative and quantitative analysis of immunoreactivity with the monoclonal antibody OX-42, which recognises the complement receptor CR3. The regions examined included the gracile nucleus, the column of Clarke and the spinal cord dorsal horn (superficial and deep laminae separately) after unilateral sciatic nerve transection, and the spinal trigeminal nucleus following unilateral infraorbital nerve transection. In all territories examined a qualitative increase in OX-42 immunoreactivity was observed 24 h postlesion. Further, quantitative analysis revealed an exponential development of the OX-42 immunoreactivity, with a peak at one week postlesion, thereafter showing a slow exponential decline. Our results show that the signal (or signals) that induces the microglial cell response in primary sensory projection territories is rapid in comparison to previously described central degenerative changes following peripheral nerve lesions (transganglionic degeneration). These findings are compatible with the hypothesis that activated microglia play a pathogenetic role in the development of transganglionic degeneration.

Regrowth of lesioned dorsal root nerve fibers into the spinal cord of neonatal rats

In postnatal rat pups the L4 and L5 dorsal roots were lesioned. After 3-6 months the spinal cord of the rats was subjected to tracing studies of regenerated dorsal root axons with transganglionically transported horseradish peroxidase (HRP) and immunohistochemistry with antibodies to calcitonin gene-related peptide (CGRP). In rats operated at birth (0-2 days old) HRP-filled profiles as well as CGRP staining were found in the outer lamina of the spinal cord dorsal horn. Signs of dorsal root nerve fiber regrowth in the spinal cord could not be found in rats which had been operated at the end of the first postnatal week or later.

The response of central glia to peripheral nerve injury

Microglial and astroglial cells undergo prompt responses to peripheral motor and sensory axon injury. These responses include proliferation of microglial cells as well as hypertrophy and increased levels of glial fibrillary acidic protein around the axotomized motoneurons and in the central projection territories of peripherally axotomized sensory ganglion cells. Proliferating microglial cells migrate towards reacting motoneurons, however, without directly apposing their cell membrane. Astroglial cells, on the other hand, increase their structural interrelationship with reacting motoneurons, seemingly at the expense of some presynaptic terminals. In sensory projection areas, microglial cells phagocytose degenerating axons and terminals. Beyond these observations, the functional role of the central glial cell response to peripheral nerve injury is obscure.

Distribution of c-fos expressing dorsal horn neurons after electrical stimulation of low threshold sensory fibers in the chronically injured sciatic nerve.

The distribution of proto-oncogene c-Fos protein-immunoreactive cells in the spinal cord dorsal horn was studied after electrical stimulation at A alpha/A beta-fiber intensity of normal and previously injured sciatic nerves in urethane anesthetized rats. No or only occasional Fos protein-like immunoreactive cells were seen after stimulation of the normal uninjured nerve or after nerve transection without stimulation. Electrical nerve stimulation at 3, 12, and 21 days after sciatic nerve transection resulted in substantial increases in the numbers of Fos protein-like immunoreactive cell nuclei in each of Rexeds laminae I-V. Combined demonstration of Fos protein-like immunoreactivity and of glial fibrillary acidic protein-like immunoreactivity (astroglia) or OX-42 immunoreactivity (microglia), indicated that the observed Fos protein-like response was confined to neurons and not to astroglia or microglia. Combined demonstration in the spinal cord of Fos protein-like immunoreactive neurons and neurons labeled retrogradely with Fluoro-Gold from the gracile nucleus showed that some of the Fos protein-like immunoreactive neurons in Rexeds laminae III and IV contributed to the postsynaptic dorsal column pathway. The results indicate that stimulation at A alpha/A beta-fiber intensity of a previously injured nerve gives rise to an abnormally increased activation pattern of postsynaptic neurons in the dorsal horn, some of which contribute to the postsynaptic dorsal column pathway.

On the use of fast blue, fluoro-gold and diamidino yellow for retrograde tracing after peripheral nerve injury: uptake, fading, dye interactions, and toxicity.

The usefulness of three retrograde fluorescent dyes for tracing injured peripheral axons was investigated. The rat sciatic was transected bilaterally and the proximal end briefly exposed to either Fast Blue (FB), Fluoro-Gold (FG) or to Diamidino Yellow (DY) on the right side, and to saline on the left side, respectively. The nerves were then resutured and allowed to regenerate. Electrophysiological tests 3 months later showed similar latencies and amplitudes of evoked muscle and nerve action potentials between tracer groups. The nerves were then cut distal to the original injury and exposed to a second (different) dye. Five days later, retrogradely labelled neurones were counted in the dorsal root ganglia (DRGs) and spinal cord ventral horn. The number of neurones labelled by the first tracer was similar for all three dyes in the DRG and ventral horn except for FG, which labelled fewer motoneurones. When used as second tracer, DY labelled fewer neurones than FG and FB in some experimental situations. The total number of neurones labelled by the first and/or second tracer was reduced by about 30% compared with controls. The contributions of cell death as well as different optional tracer combinations for studies of nerve regeneration are discussed.

Distribution of antinociceptive adenosine A1 receptors in the spinal cord dorsal horn, and relationship to primary afferents and neuronal subpopulations.

Adenosine can reduce pain and allodynia in animals and man, probably via spinal adenosine A1 receptors. In the present study, we investigate the distribution of the adenosine A1 receptor in the rat spinal cord dorsal horn using immunohistochemistry, in situ hybridization, radioligand binding, and confocal microscopy. In the lumbar cord dorsal horn, dense immunoreactivity was seen in the inner part of lamina II. This was unaltered by dorsal root section or thoracic cord hemisection. Confocal microscopy of the dorsal horn revealed close anatomical relationships but no or only minor overlap between A1 receptors and immunoreactivity for markers associated with primary afferent central endings: calcitonin gene-related peptide, or isolectin B4, or with neuronal subpopulations: mu-opioid receptor, neuronal nitric oxide synthase, met-enkephalin, parvalbumin, or protein kinase Cgamma, or with glial cells: glial fibrillary acidic protein. A few adenosine A1 receptor positive structures were double-labeled with alpha-amino-3-hydroxy-5-methyl-4-isoaxolepropionic acid glutamate receptor subunits 1 and 2/3. The results indicate that most of the adenosine A1 receptors in the dorsal horn are located in inner lamina II postsynaptic neuronal cell bodies and processes whose functional and neurochemical identity is so far unknown. Many adenosine A1 receptor positive structures are in close contact with isolectin B4 positive C-fiber primary afferents and/or postsynaptic structures containing components of importance for the modulation of nociceptive information.