Preventing regeneration of infraorbital axons does not alter the ganglionic or transganglionic consequences of neonatal transection of this trigeminal branch

Retrograde and transganglionic tracing with a combination of horseradish peroxidase (HRP) and wheatgerm agglutinin (WGA)-conjugated HRP (WGA-HRP) was employed to determine whether transection of the infraorbital (IO) nerve on the day of birth and prevention of regeneration by retransecting it at weekly intervals until the time of a terminal anatomical experiment had effects upon ganglion cell survival and innervation of the brainstem by this trigeminal (V) branch that differed from those which followed a single transection of the same nerve on the day of birth without any attempt to prevent peripheral regeneration of the cut axons. Counts of labelled ganglion cells and examination of the brainstem labelling produced by application of HRP and WGA-HRP to the IO nerve proximal to the point of transection(s) at 6 weeks of age demonstrated no differential effects of preventing regeneration of the cut nerve. In animals subjected to a single transection of the nerve (n = 9), we counted an average of 5001.2 (S.D. = 1286.9) labelled ganglion cells and these had an average diameter of 22.7 micron (S.D. = 6.3). In the rats (n = 9) that sustained multiple nerve cuts, the average number of labelled ganglion cells was 4447.8 (S.D. = 1060.9). The mean diameter for these primary afferent neurons was 21.5 micron (S.D. = 6.6). Neither of these values were significantly different from those from the rats subjected to a single nerve cut. The cell counts from both of these groups were significantly lower than those obtained after application of HRP and WGA-HRP to the IO nerve in normal rats (n = 3, X = 12,553.3, S.D. = 1454.8), but the average cell diameter in the normals (X = 23.2, S.D. = 6.6) was not significantly greater than that in the nerve-damaged animals. The pattern of brainstem labelling observed in the rats subjected to multiple nerve cuts was the same as that in the rats which sustained a single transection of the IO nerve on the day of birth. Very little terminal labelling was observed in nucleus principalis, subnucleus oralis, subnucleus interpolaris or the magnocellular portion of caudalis. There was, however, very heavy labelling in laminae I and II of the latter nucleus.     

Central projections of the rat radial nerve investigated with transganglionic degeneration and transganglionic transport of horseradish peroxidase

Transganglionic degeneration and transganglionic transport of HRP were used for investigation of the spinal cord and brainstem projections from the superficial, cutaneous (SR) and deep, muscular (DR) branches of the radial nerve. The HRP study included a numerical and size analysis of labelled dorsal root ganglion (DRG) cells. In degeneration experiments the SR nerve was found to project somatotopically to laminae III-IV, but degeneration was also found in lamina I and inconsistently in lamina II. Transection of the DR nerve was found to give rise to a small amount of degeneration, which in "sham" operations was established to result from the skin injury during dissection of the DR nerve. With the HRP method, the SR nerve was found to project somatotopically to laminae I-IV, whereas the DR nerve projected more diffusely to the medial part of laminae V-VII. HRP application to the SR and DR nerves resulted in labelling of a mean of 1,024 and 310 DRG cells, respectively. These labelled neurons had a median cell area of 381 and 562 micron 2 for the SR and DR nerves, respectively, and both small and large cells were labelled in both types of experiments. In the lower brainstem, projections from the SR nerve were found only in the ipsilateral dorsal part of the main cuneate nucleus (MCN) with both methods. Brainstem projections from the DR nerve that were found only with the HRP method were found in the ipsilateral ventral part of the MCN together with a projection to the ipsilateral external cuneate nucleus. No projections were found to the central cervical nucleus. The present results indicate that cutaneous compared to muscular primary sensory neurons are much more prone to react with transganglionic degeneration after peripheral nerve transection. Furthermore, in the rat the SR nerve projects somatotopically, whereas the DR nerve does not. Both nerve branches are connected to small and large spinal ganglion cells, although the median cell area is larger in muscular neurons.     

Increase of preprotachykinin mRNA and substance P immunoreactivity in spared dorsal root ganglion neurons following partial sciatic nerve injury

Complete sciatic nerve injury reduces substance P (SP) expression in primary sensory neurons of the L4 and L5 dorsal root ganglia (DRG), due to loss of target-derived nerve growth factor (NGF). Partial nerve injury spares a proportion of DRG neurons, whose axons lie in the partially degenerating nerve, and are exposed to elevated NGF levels from Schwann and other endoneurial cells involved in Wallerian degeneration. To test the hypothesis that SP is elevated in spared DRG neurons following partial nerve injury, we compared the effects of complete sciatic nerve transection (CSNT) with those of two types of partial injury, partial sciatic nerve transection (PSNT) and chronic constriction injury (CCI). As expected, a CSNT profoundly decreased SP expression at 4 and 14 days postinjury, but after PSNT and CCI the levels of preprotachykinin (PPT) mRNA, assessed by in situ hybridization, and the SP immunoreactivity (SP-IR) of the L4 and L5 DRGs did not decrease, nor did dorsal horn SP-IR decrease. Using retrograde labelling with fluorogold to identify spared DRG neurons, we found that the proportion of these neurons expressing SP-IR 14 days after injury was much higher than in neurons of normal DRGs. Further, the highest levels of SP-IR in individual neurons were detected in ipsilateral L4 and L5 DRG neurons after PSNT and CCI. We conclude that partial sciatic nerve injury elevates SP levels in spared DRG neurons. This phenomenon might be involved in the development of neuropathic pain, which commonly follows partial nerve injury.     

Anterograde transsynaptic transport of WGA-HRP from spinal afferents to postganglionic sympathetic cells of the stellate ganglion of the guinea pig.

After injection of wheat germ agglutinin conjugated horseradish peroxidase (WGA-HRP) or choleragenoid conjugated HRP (B-HRP) into lower cervical and upper thoracic dorsal root ganglia (DRG), HRP reaction product was observed in peripheral fibers of spinal afferents and in postganglionic cell bodies of the stellate ganglion (SG) in the guinea pig. After WGA-HRP injection into C8-T3 or T5 DRG, HRP-labelled cells were observed to cluster at the rami within the SG, with peak labelling observed 36 h after injection. SG cell labelling occurred with B-HRP as well, but not with native HRP after injection into thoracic DRG. Injection of this tracer in C8 DRG gave rise to a small number of labelled cells. In contrast to the labelling pattern following thoracic or C8 DRG injections, injection of WGA-HRP or native HRP into C6 DRG, led to random SG cell labelling. We conclude that the anterograde transsynaptic transport, following injection of WGA-HRP into thoracic DRG, provides a method to selectively label a population of postganglionic sympathetic neurons within the SG. A combination of transsynaptic and retrograde transport appears to be responsible for labelling after injection into C8 DRG, whereas labelling after C6 DRG injections seems to be due primarily to retrograde transport.     

Distribution of transganglionically labelled soybean agglutinin primary afferent fibres after nerve injury

The distribution of the retrogradely-transganglionically transported lectin soybean agglutinin (SBA) and of SBA conjugated to horseradish peroxidase (SBA–HRP) has been examined in the L_4–5 dorsal root ganglia, lumbar spinal cord and gracile nucleus at 2, 6 and 14 weeks after sciatic nerve transection and ligation. Cell size analysis showed there were no changes in the mean area of labelled DRG profiles after injury. In the spinal cord, terminal labelling was restricted to laminae I and II with no evidence of labelling in novel territories such as the deeper laminae after injury. At 2 weeks, the labelling on the injured side was similar in distribution and intensity to that of the contralateral, uninjured side. At 6–14 weeks the labelling on the injured side was significantly weaker as compared to the contralateral side, but not completely depleted. In the gracile nucleus, at all survival times, an increased distribution and amount of labelling was seen which may reflect sprouting of C and A-delta fibres. These results suggest that SBA is a useful tracer to study the effects of nerve injury on the central terminals of axotomised afferents terminating in laminae I–II and that C-fibres appear not to sprout outside their normal laminar distribution in the dorsal horn after injury.     

Sensory neurons of the rat sciatic nerve

Experiments have been undertaken in this laboratory over recent years to accurately determine the numbers and sizes of somatic neurons which contribute to the normal sciatic nerve, at mid-thigh levels, of the adult, albino rat. This article is concerned with the dorsal root ganglion (DRG) neuron population of the sciatic nerve whose cell bodies were identified through retrograde labeling of cut branches of the sciatic with horseradish peroxidase (HRP) and/or its wheat germ conjugate (WGA-HRP). It is essential to understand the neuronal composition of the normal rat sciatic nerve if the consequences of aging, nerve injury, and surgical repair to improve functional regeneration are to be properly evaluated. Neuron counts were determined from camera-lucida paper drawings of all labeled profiles in DRGs L3-L6 at 100 x magnification. The profiles, obtained by labeling individual branches of the sciatic nerve (sural, lateral sural, tibial, peroneal, medial, and lateral gastrocnemius/soleus nerves) were traced from 40-microns-thick, serial, frozen sections. The sizes of the perikarya, areas and diameters, were determined by tracing the perimeters of the drawn profiles on a digitizing tablet. The tablet's output was inputted directly into a specially designed computer spreadsheet which contained a mathematical table for correcting the split-cell error inherent to the sectioning process. Afferents from any given branch of the sciatic normally occupied two to three adjacent ganglia. Sciatic DRG neurons were normally located in lumbar ganglia L3-L6. Nearly 98-99% of all sciatic DRG perikarya resided in the L4 and L5 DRGs. The L6 DRG, traditionally regarded as an important contributor to the rat sciatic, contained merely 0.4% of its afferent neurons while the L3 ganglion, frequently overlooked as a contributor, contained 1.2% of the mid-thigh sciatic afferents. The mean size of rat DRG neurons was about 29 microns (550-600 microns2). The corrected counts revealed that the normal sciatic nerve (at mid-thigh levels), in rats between 2 and 12 months of age, contained a mean, total DRG neuron population of about 10,500 neurons. This is probably an underestimate by 3-5% of the true number due to occasional unreliable labeling of some of the small DRG neurons. It is estimated that the normal, mean number of sciatic DRG neurons of young to middle-aged rats lies somewhere between 10,500 and 11,000 +/- 2000. The data suggest that nearly 20% of all DRG neurons in the sciatic nerve supply muscle afferents. The vast majority of the remaining neurons are involved with innervation of the skin.(ABSTRACT TRUNCATED AT 400 WORDS)     

Anterograde transport of horseradish-peroxidase conjugated isolectin B4 from Griffonia simplicifolia I in spinal primary sensory neurons of the rat

Anterograde transport of the isolectin B4 from Griffonia simplicifolia I (B4) conjugated to horseradish peroxidase (HRP) was investigated in rat somatic and visceral primary sensory neurons at different spinal levels. Injection of B4-HRP into the L5 dorsal root ganglion (DRG) resulted in labelling in the sural nerve, but not in the gastrocnemius nerves. Free nerve endings and lanceolate-like nerve endings were labelled in the lateral hindpaw skin. Labelled fibres were also observed in the greater splanchnic nerve following B4-HRP injection into the T10–11 DRGs. Electron microscopic examination of the labelled nerves showed that B4-HRP labelled exclusively unmyelinated axons. In the spinal cord, labelling was observed in the superficial dorsal horn, and additionally, although much more sparse, in the medial and lateral collateral projections following injections into the T10–11 DRGs. The results suggest that B4-HRP should be a suitable anterograde tracer of unmyelinated cutaneous and splanchnic but not muscle primary afferent fibres.     

Selective reinnervation of somatotopically appropriate muscles after facial nerve transection and regeneration in the neonatal rat

The organization of the facial motor nucleus (FMN) has been examined after transection and regeneration of the facial nerve (FN) in neonatal and adult rats. In one series of experiments, horseradish peroxidase (HRP) was applied bilaterally to the superior or inferior buccal ramus 5 months after neonatal FN transection. In another series of experiments, wheat germ agglutinin-horseradish peroxidase conjugate was injected in selected vibrissae follicular muscles on both sides in animals surviving 5 months after FN transection at the neonatal or adult stage. The number and distribution of HRP-labeled cell bodies in the FMN after regeneration was compared with the contralateral side. On the uninjured side, labeled neurons were somatotopically organized. Ipsilateral to nerve injury the number of labeled cells was markedly reduced after neonatal nerve transection, but somatotopy was preserved. However, after nerve lesion at the adult stage, no significant loss of motoneurons occurred, but motor nucleus somatotopy was not maintained. Two alternative principal explanations are proposed for the re-establishment of the normal somatotopy after neonatal injury: that regenerating axons grow in a random fashion but inappropriate connections are subsequently eliminated or that regenerating axons of surviving neurons immediately follow a pathway leading to the appropriate muscle.     

Axonal injury-dependent induction of the peripheral benzodiazepine receptor in small-diameter adult rat primary sensory neurons

The peripheral benzodiazepine receptor (PBR), a benzodiazepine but not γ‐aminobutyric acid‐binding mitochondrial membrane protein, has roles in steroid production, energy metabolism, cell survival and growth. PBR expression in the nervous system has been reported in non‐neuronal glial and immune cells. We now show expression of both PBR mRNA and protein, and the appearance of binding of a synthetic ligand, [³H]PK11195, in dorsal root ganglion (DRG) neurons following injury to the sciatic nerve. In naïve animals, PBR mRNA, protein expression and ligand binding are undetectable in the DRG. Three days after sciatic nerve transection, however, PBR mRNA begins to be expressed in injured neurons, and 4 weeks after the injury, expression and ligand binding are present in 35% of L4 DRG neurons. PBR ligand binding also appears after injury in the superficial dorsal horn of the spinal cord. The PBR expression in the DRG is restricted to small and medium‐sized neurons and returns to naïve levels if the injured peripheral axons are allowed to regrow and reinnervate targets. No non‐neuronal PBR expression is detected, unlike its putative endogenous ligand the diazepam binding inhibitor (DBI), which is expressed only in non‐neuronal cells, including the satellite cells that surround DRG neurons. DBI expression does not change with sciatic nerve transection. PBR acting on small‐calibre neurons could play a role in the adaptive survival and growth responses of these cells to injury of their axons.     

Kinetics of production of a novel growth factor after peripheral nerve injury

In response to transection injury, the distal segment of sciatic nerve produces a soluble factor which stimulates neurite outgrowth from 15 day embryonic rat dorsal root ganglion (DRG) neurons, and PC12 cells. This activity enhances survival of large sensory neurons, promotes myelination and has been designated SN. The expression of SN, undetectable in the perineurium and proximal segments, occurs solely in the endoneurium distal to the site of permanent transection. When the distal portion is removed immediately after transection, homogenized and the supernatant tested, there is little neurite promoting activity in the normal nerve. For the first 10 days after transection the major soluble factor present in the distal segment is NGF. The amount of neurite promoting activity increases after 10 days and appears to plateau at 30-35 days while the proportion that is inhibited by anti-NGF decreases. In a competitive receptor binding assay, SN does not compete with 125I-NGF for receptors on either DRG or PC12 cells. Separation using polyacrylamide-agarose followed by HPLC demonstrates that SN migrates with polypeptides of molecular weights 17.2 and 19.1 kDa.     

Effect of fetal infraorbital nerve transection upon trigeminal primary afferent projections in the rat

Transganglionic tracing with a combination of horseradish peroxidase (HRP) and wheat germ agglutinin-conjugated HRP (WGA-HRP) was employed to compare the trigeminal (V) innervation of the brainstem in adult rats that sustained transection of the infraorbital nerve (ION) on either the day of birth or just prior to the beginning of the 17th embryonic day (E-17). The same methods were also employed to assess the effects of such lesions upon the innervation of the brainstem by the lingual, inferior alveolar, mylohyoid, and auriculotemporal V branches. Previous experiments (Chiaia et al.: Dev. Brain Res. 36:75-88, '87) showed that application of HRP and WGA-HRP to the ION in normal adult rats (N = 3) labelled 12,553 +/- 1,455 (mean +/- s.d.) V ganglion cells while application of these tracers to the regenerated ION after neonatal transection (N = 9) labelled 5,001 +/- 1,287 ganglion cells. Application of HRP and WGA-HRP to the regenerated ION in adulthood (N = 6) after fetal transection labelled 5,476 +/- 3,056 ganglion cells. Thus, the numbers of ganglion cells giving rise to the regenerated ION after fetal and neonatal transection were equivalent (P greater than .05). The central projections of the ION after fetal transection were qualitatively different from those observed after neonatal injury. After neonatal transection, the central terminal field of regenerated ION fibers in adulthood is almost completely restricted to layers I and II of subnucleus caudalis (SpC; Jacquin and Rhoades: Brain Res. 269:137-144, '83; Chiaia et al.: Dev. Brain Res. 36:75-88, '87). After fetal transection, regenerated ION axons terminate heavily in all portions of the V brainstem complex. After neonatal ION transection, we (Jacquin and Rhoades: J. Comp. Neurol. 235:129-143, '85) have been unable to detect central sprouting of undamaged V mandibular axons by means of transganglionic tracing with HRP and WGA-HRP. Such sprouting was evident in both V subnucleus interpolaris (SpI) and SpC after fetal ION transection. We carried out one additional experiment to determine whether ION ganglion cells that survived fetal axotomy were more resistant to axonal damage than the population of neurons that normally contribute to this nerve on the day of birth. Rats (N = 5) sustained transection of the ION on E-17 and again on the day of birth. The regenerated ION was then labelled with HRP and WGA-HRP when the animals reached adulthood.(ABSTRACT TRUNCATED AT 400 WORDS)     

Directional specificity of regenerating primary sensory neurons after peripheral nerve crush or transection and epineurial suture A sequential double-labeling study in the rat

Clinical and experimental observations have demonstrated that peripheral nerve transection generally results in lasting disturbed sensory discrimination whereas nerve crush is followed by more or less complete functional restoration. This has been explained by an increased misdirection of regenerating fibers after transection as compared to crush injury. In the present study, sequential double-labeling was used to investigate the relative proportions of peripherally misdirected sensory fibers in the sural and tibial nerve branches after crush or transection of the parent sciatic nerve in the rat. Control experiments showed that 0.21% ± 0.12 (mean ± S.D.) of all labeled tibial and sural neurons normally send axons to both nerves. After sciatic nerve crush or transection, 1.31% ± 0.78 and 3.79% ± 3.01, respectively, of all labeled tibial and sural axons were double-labeled indicating previously sural axons now having an axon in the tibial. Statistically significant differences in the percentages of bidirectional sciatic sensory neurons were found between the normal controls and after crush injury (P < 0.01) or transection injury (P < 0.001), respectively, but not between transection and crush (P > 0.05). The results indicate that the number of sensory neurons having an axon in two peripheral nerves is normally very small, that a substantial number of sensory axons become misdirected after both crush and transection with resuture, and that the number of misdirected fibers in the major sciatic branches after these types of injury is similar.     

Retrograde axonal transport of horseradish peroxidase from cornea to trigeminal ganglion

Summary Horseradish peroxidase (HRP) was dripped on the scarified left cornea of adult mice. Twentyfour hours later the animals were fixed by vascular perfusion and frozen sections cut from both trigeminal ganglia.After incubation for peroxidase activity labelled nerve cells were restricted to the medial ophthalmic part of the ganglion ipsilateral to HRP administration. If the scarification was omitted no neuronal labelling was observed. This labelling of the neurons is most probably the result from axonal uptake and subsequent retrograde axonal transport of the tracer.The similarity in distribution of peroxidase labelled nerve cells and the first ganglionic lesions occurring after instillation of herpes simplex virus in the cornea is pointed out.     

Dynamic changes in glypican-1 expression in dorsal root ganglion neurons after peripheral and central axonal injury

Glypican-1, a glycosyl phosphatidyl inositol (GPI)-anchored heparan sulphate proteoglycan expressed in the developing and mature cells of the central nervous system, acts as a coreceptor for diverse ligands, including slit axonal guidance proteins, fibroblast growth factors and laminin. We have examined its expression in primary sensory dorsal root ganglion (DRG) neurons and spinal cord after axonal injury. In noninjured rats, glypican-1 mRNA and protein are constitutively expressed at low levels in lumbar DRGs. Sciatic nerve transection results in a two-fold increase in mRNA and protein expression. High glypican-1 expression persists until the injured axons reinnervate their peripheral targets, as in the case of a crushed nerve. Injury to the central axons of DRG neurons by either a dorsal column injury or a dorsal root transection also up-regulates glypican-1, a feature that differs from most DRG axonal injury-induced genes, whose regulation changes only after peripheral and not central axonal injury. After axonal injury, the cellular localization of glypican-1 changes from a nuclear pattern restricted to neurons in noninjured DRGs, to the cytoplasm and membrane of injured neurons, as well as neighbouring non-neuronal cells. Sciatic nerve transection also leads to an accumulation of glypican-1 in the proximal nerve segment of injured axons. Glypican-1 is coexpressed with robo 2 and its up-regulation after axonal injury may contribute to an altered sensitivity to axonal growth or guidance cues.     

Sciatic nerve regeneration across gaps within silicone chambers: long-term effects of NGF and consideration of axonal branching

We examined whether the short-term beneficial effects of nerve growth factor (NGF) upon regeneration are sustained over a prolonged period of time across 8-mm gaps within silicone chambers. Rat sciatic nerve regeneration both with and without NGF was examined after 10 weeks. Myelinated counts from the regenerated sciatic and distal tributary nerves were correlated to the numbers of motor and sensory neurons retrogradely labeled with horseradish peroxidase (HRP) applied distal to the regenerated segment. Regenerated sciatic and sural nerves were examined ultrastructurally for morphological analysis. Both regenerated groups by 10 weeks achieved essentially complete counts of myelinated axons in the distal tributary nerves and the regenerated segment of the sciatic nerve compared to the uninjured controls. There were similar numbers of retrogradely labeled sensory and motor neurons in the dorsal root ganglia (DRG) and lumbar spinal cord of both groups and, surprisingly, of the uninjured normal control group. Ultrastructural analysis demonstrated no difference in the distribution of axonal diameters or myelin thickness between the regenerated groups. In evaluating regeneration in experimental silicone chamber models, it is important to determine such parameters as the percentage of neurons that grow across the gap and the incidence of axonal sprouting. One can then make accurate assessments of experimental perturbations and predict whether they improve the naturally occurring regeneration through chambers. These results must ultimately be compared with equivalent determinations in the uninjured nerve. At 10 weeks there was essentially complete regeneration of both the NGF and control regenerative groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect on the rat hypoglossal nucleus of vinblastine and colchicine applied to the intact or transected hypoglossal nerve

The possibility that interruption of axonal transport in otherwise intact axons induces retrograde neuronal and nonneuronal reactions was examined. In addition, the proposal that blockade of axonal transport proximal to nerve injury might inhibit or delay the axon reaction was examined. Cuffs containing various doses of vinblastine were applied to the intact hypoglossal nerve. Colchicine was applied in a similar way to the intact hypoglossal nerve, injected directly into the intact nerve, or administered proximal to the site of hypoglossal nerve transection. The effect on retrograde axonal transport in the nerve was evaluated in the vinblastine experiments by the retrograde horseradish peroxidase (HRP) technique following injection of HRP or wheat germ agglutinin-conjugated HRP into the tongue. A dose of 0.01% caused an almost complete, but transient, blockade of the retrograde transport of the tracer, and induced a clearcut chromatolytic reaction in hypoglossal neurons. The chromatolytic changes were accompanied by a significant increase in the number of glial cells, many of which were identified as microglia. Similar results were obtained with colchicine alone or in combination with nerve transection. Signs of Wallerian degeneration after vinblastine treatment (0.01%) were observed only in a small number of myelinated fibers. The findings are compatible with the view that depletion of retrogradely transported factors from the peripheral innervation territory (including the distal nerve stump) to the perikaryon and/or a premature return of anterogradely transported substances at the site of drug exposure are factors inducing retrograde neuronal and nonneuronal changes.     

The organization of visceral sensory neurons in thoracic dorsal root gangla (DRG) of the cat studied by horseradish peroxidase (HRP) reaction using the cryostat

The organization of visceral sensory neurons in thoracic dorsal root ganglia (DRG) was studied by retrograde transport of horseradish peroxidase (HRP) from the central cut end of the left major splanchnic nerve of the cat. The majority of HRP-labeled cells were concentrated between T5 and T11. Within a DRG, labeled splanchnic neurons were found in all sectors. There was no consistent pattern of localization within the ganglion although clustering of visceral cell bodies was apparent. It may be that each clustered group of cells innervates individual viscera or reflects a degree of functional segregation.     

Prepro-VIP and preprotachykinin mRNAs in the rat dorsal root ganglion cells following peripheral axotomy

Using in situ hybridization histochemistry, we examined the expression of prepro-vasoactive intestinal polypeptide (VIP) mRNAs and preprotachykinin (PPT) mRNAs which coded for substance P (SP) in the rat dorsal root ganglion (DRG) following spinal nerve transection. VIP mRNAs increased dramatically in the DRG neurons after transection of the peripheral branch of the spinal nerve (sciatic nerve), whereas PPT mRNAs showed a gradual decrease for a few weeks. Dorsal rhizotomy or axotomy of the central branch of DRG cells had little influence on VIP-mRNAs and no effect on PPT mRNA expression. These results demonstrated an activation of VIP biosynthesis in the DRG neurons due to axotomy of the peripheral branch, which was opposite to the reaction of PPT mRNA to the same treatment.     

Comparison of c-jun,junB, and junD mRNA expression and protein in the rat dorsal root ganglia following sciatic nerve transection

The present study was designed to compare the expression of the Jun family of protooncogenes following nerve injury. Adult rats were anesthetized and the sciatic nerve transected. Dorsal root ganglia (DRG) at 1, 2, 3, and 7 days after nerve transection were collected, their total RNA extracted, and Northern blots performed using ³²P-labeled oligonucleotide probes. The constitutive expression of c-jun mRNA was very low in DRG. Induction of c jun mRNA was observed by day 1 after nerve transection, with a sixfold peak at 3 stays and a twofold induction still present by day 7. The constitutive expression of junB mRNA was also low in the DRG, and sciatic nerve transection produced only a modest induction (1.7fold by day 3) in the DRG ipsilateral to the nerve cut. junD mRNA was constitutively expressed at high levels in the DRG, and its level of expression did not change after sciatic nerve transection. Immunocytochemistry studies demonstrated a pattern of c-Jun, JunB, and JunD immunoreactivity (IR) associated with the cell nuclei of DRG neurons. c-Jun IR was found at very low levels in the undamaged contralateral DRG neurons, but sciatic nerve transection dramatically increased the number of c-Jun-immunoreactive neurons. Dot blot immunoblotting assay confirmed that the DRG ipsilateral to the sciatic nerve cut contained a higher level of c-Jun protein than the contralateral control DRG. Similar to c-Jun IR, JunB IR was minimal in the undamaged contralateral DRAG. However, the DRG ipsilateral to the nerve transection did not show an increase in the number of immunoreactive neurons. JunD protein was expressed at high levels in the contralateral DRG, and this level of expression persisted after sciatic nerve transection in the ipsilateral DRG. DNA gel retardation assay experiments with an AP-1 consensus sequence showed a single DNA-protein complex. This complex was increased in ipsilateral as compared with contralateral DRG extracts. The amount of DNA protein complex was reduced byc-Jun protein antiserum but was not altered when treated with a Fos antibody. We conclude that cjun, junB and junD mRNAs and proteins are differentially regulated in the DRG after sciatic nerve transection. © 1995 Wiley-Liss, Inc.