Differential expression of galanin immunoreactivities in the primary sensory neurons following partial and complete sciatic nerve injuries

Neuropeptide expression in primary sensory neurons is highly plastic in response to peripheral nerve axotomy. While neuropeptide changes following complete sciatic nerve injury have been extensively studied, much less is known about the effects of partial sciatic nerve injuries on neuropeptide plasticity. Galanin. a possible endogenous analgesic peptide, was up-regulated in primary sensory neurons following complete sciatic nerve injury. We investigated the effects of partial sciatic nerve injuries on galanin expression in primary sensory neurons, and compared this effect with that after complete sciatic nerve injury. Complete transection, partial transection and chronic constriction injury were made, respectively, on the sciatic nerves of three groups of rats at high thigh level. Animals were allowed to survive for four and 14 days before being killed. L4 and L5 dorsal root ganglia, L4 5 spinal cord and lower brainstem were processed for galanin immunocytochemical staining. After all three types of sciatic nerve injuries, galanin-immunoreactive neurons were significantly increased in the ipsilateral dorsal root ganglia, and galanin-immunoreactive axonal fibres were dramatically increased in the superficial laminae of the dorsal horn and the gracile nuclei, compared to the contralateral side. However, in partial injury models, the percentages of galanin-immunoreactive dorsal root ganglion neurons were significantly higher than in complete nerve transection. Size frequency distribution analysis detected that more medium- and large-size galanin-immunoreactive dorsal root ganglion neurons were present after partial nerve transection and constriction injury than after complete nerve transection. Using a combined approach of retrograde tracing of flurorescent dyes and galanin immunostaining, we found that a partial transection increased the proportions of galanin-immunoreactive neurons among both axotomized and non-axotomized neurons. Galanin-immunoreactive axonal fibres were not only detected in the superficial laminae, but also in the deeper laminae of the dorsal horn of partial injury animals. Furthermore, more galanin-immunoreactive axonal fibres were observed in the ipsilateral gracile nuclei of partially injured rats than in completely injured rats. We conclude that partial sciatic nerve injuries induced greater galanin up-regulation in medium- and large-size dorsal root ganglion neurons than complete sciatic nerve injury. Galanin expression in primary sensory neurons seems to be differentially regulated following partial and complete sciatic nerve injuries.     

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.     

Partial and complete sciatic nerve injuries induce similar increases of neuropeptide Y and vasoactive intestinal peptide immunoreactivities in primary sensory neurons and their central projections

Partial nerve injury is more likely to cause neuropathic pain than complete nerve injury. We have compared the changes in neuropeptide expression in primary sensory neurons which follow complete and partial injuries to determine if these might be involved. Since more neurons are damaged by complete injury, we expected that complete sciatic nerve injury would simply cause greater increases in neuropeptide Y and vasoactive intestinal peptide than partial injury. We examined neuropeptide Y and vasoactive intestinal peptide immunoreactivities in L4 and L5 dorsal root ganglia, the dorsal horn of L4-L5 spinal cord, and the gracile nuclei of rats killed 14 days after unilateral complete sciatic nerve transection, partial sciatic nerve transection and chronic constriction injury of the sciatic nerves. In all three groups of rats, neuropeptide Y- and vasoactive intestinal peptide-immunoreactive neurons were increased in the ipsilateral L4 and L5 dorsal root ganglion when compared with the contralateral side. Most neuropeptide Y-immunoreactive neurons were of medium and large size, but a few were small. Neuropeptide Y-immunoreactive axonal fibers were increased from laminae I to IV, and vasoactive intestinal peptide-immunoreactive axonal fibers were increased in laminae I and II, of the ipsilateral dorsal horn of L4-L5 spinal cord. The increases of neuropeptide Y and vasoactive intestinal peptide immunoreactivities in the dorsal horn were similar among the three groups. However, only after constriction injury were some vasoactive intestinal peptide-immunoreactive neurons seen in the deeper laminae of the ipsilateral dorsal horn. Robust neuropeptide Y-immunoreactive axonal fibers and some neuropeptide Y-immunoreactive cells were seen in the ipsilateral gracile nuclei of all three groups of animals, but neuropeptide Y-immunoreactive cells were more prominent after constriction injury. Contrary to our expectations, partial and complete sciatic nerve injuries induced similar increases in neuropeptide Y and vasoactive intestinal peptide in lumbar dorsal root ganglion neurons and their central projections in the dorsal horn and the gracile nuclei two weeks after injury. Some neurons whose axons were spared by partial injury may also increase neuropeptide Y or vasoactive intestinal peptide expression. Altered neuropeptide release from these functional sensory neurons may play a role in neuropathic pain.     

Neuronal injury increases retrograde axonal transport of the neurotrophins to spinal sensory neurons and motor neurons via multiple receptor mechanisms

We investigated the retrograde axonal transport of 125I-labeled neurotrophins (NGF, BDNF, NT-3, and NT-4) from the sciatic nerve to dorsal root ganglion (DRG) sensory neurons and spinal motor neurons in normal rats or after neuronal injury. DRG neurons showed increased transport of all neurotrophins following crush injury to the sciatic nerve. This was maximal 1 day after sciatic nerve crush and returned to control levels after 7 days. 125I-BDNF transport from sciatic nerve was elevated with injection either proximal to the lesion or directly into the crush site and after transection of the dorsal roots. All neurotrophin transport was receptor-mediated and consistent with neurotrophin binding to the low-affinity neurotrophin receptor (LNR) or Trk receptors. However, transport of 125I-labeled wheat germ agglutinin also increased 1 day after sciatic nerve crush, showing that increased uptake and transport is a generalized response to injury in DRG sensory neurons. Spinal cord motor neurons also showed increased neurotrophin transport following sciatic nerve injury, although this was maximal after 3 days. The transport of 125I-NGF depended on the expression of LNR by injured motor neurons, as demonstrated by competition experiments with unlabeled neurotrophins. The absence of TrkA in normal motor neurons or after axotomy was confirmed by immunostaining and in situ hybridization. Thus, increased transport of neurotrophic factors after neuronal injury is due to multiple receptor-mediated mechanisms including general increases in axonal transport capacity.     

Changes in brain-derived neurotrophic factor immunoreactivity in rat dorsal root ganglia, spinal cord, and gracile nuclei following cut or crush injuries

In the present study, we evaluated changes in brain-derived neurotrophic factor (BDNF) immunoreactivity in the rat lumbar (L) 5 dorsal root ganglion (DRG) and areas where afferents from the DRG terminate, the L5 spinal cord and gracile nuclei, following unilateral sciatic nerve transection or crush. From 3 days to 4 weeks following cut or crush injury, the percentage of medium and large BDNF-immunoreactive neurons in the ipsilateral DRG increased significantly compared with those on the contralateral side. Following cut injury, there was no significant change in the percentage of small BDNF-immunoreactive neurons in the ipsilateral DRG; however, the intensity of immunoreactivity of these cells decreased. Following crush injury, however, both the percentage and intensity of small BDNF-immunoreactive neurons in the ipsilateral DRG significantly increased. Following cut injury, the expression of BDNF-immunoreactive axonal fibers decreased markedly in the ipsilateral superficial laminae of the L5 spinal cord and increased significantly in the ipsilateral deeper laminae of the spinal cord and gracile nuclei. Crush injury induced a marked increase in the expression of BDNF-immunoreactive axonal fibers in the superficial laminae of the spinal cord and gracile nuclei. These differences in BDNF response in the DRG and spinal cord after cut or crush injuries may reflect differences in trophic support to the injured DRG neurons and altered neuronal activity in the spinal cord and gracile nuclei following different types of peripheral nerve injury.     

Motor denervation induces altered muscle fibre type densities and atrophy in a rat model of neuropathic pain

Loose ligation of a sciatic nerve in rats (chronic constriction injury; CCI) provokes sensory, autonomic, and motor disturbances like those observed in humans with partial peripheral nerve injury. So far, it is unknown whether these motor disturbances result from (mechanical) allodynia or from damage to the motor neuron. These considerations prompted us to assess, in CCI rats, the density of motor axons in both the ligated sciatic nerve and the ipsilateral femoral nerve. To this end, we determined the number of cholinesterase positive fibres. It has been demonstrated previously that muscle fibre type density may be used as a measure of motor denervation and/or hypokinesia. Therefore, the myofibrillar ATPase reaction was employed to assess fibre type density in biopsies obtained from the lateral gastrocnemius muscle (innervated by sciatic nerve) and rectus femoris muscle (innervated by femoral nerve). We observed axonal degeneration of motor fibres within the loosely ligated sciatic nerve, both at an intermediate (day 21) and at a late stage (day 90) after nerve injury. The reduction in the number of motor nerve fibres was more pronounced distal to the site of the ligatures than proximal. A (less pronounced) reduction of motor fibres was observed in the ipsilateral (non-ligated) femoral nerve. In line with these findings, we observed altered fibre type densities in muscle tissue innervated by the ligated sciatic nerve as well as the non-ligated femoral nerve indicative of motor denervation rather than hypokinesia. The findings of this study suggest that the motor disorder induced by partial nerve injury involves degeneration of motor nerve fibres not only within the primarily affected nerve but also within adjacent large peripheral nerves. This spread outside the territory of the primarily affected nerve suggests degeneration of motor neurons at the level of the central nervous system.     

Islet amyloid polypeptide and calcitonin gene-related peptide expression are down-regulated in dorsal root ganglia upon sciatic nerve transection

Islet amyloid polypeptide (IAPP) is structurally related to calcitonin gene-related peptide (CGRP) and has been implicated in glucose homeostasis and diabetes pathogenesis because it is expressed in insulin cells and forms amyloid in pancreatic islets from type II diabetic patients. IAPP is also constitutively co-expressed with CGRP in rat sensory neurons. Whether expression of IAPP is altered by nerve injury with or without regeneration was investigated in adult rats subjected to unilateral sciatic axotomy; IAPP and CGRP expression were determined by quantitative in situ hybridization and immunocytochemistry at days 3, 10 and 30 after axotomy. In ipsilateral L4-L5 dorsal root ganglia (DRG), the percentages of nerve cell profiles labelled for IAPP and CGRP mRNA were reduced at all time points studied. IAPP and CGRP mRNA expression were lower in nerve cell profiles in ipsilateral DRGs compared to the contralateral side after axotomy alone whereas epineurial nerve suture maintained or restored IAPP and CGRP expression. The numbers of IAPP- and CGRP-immunoreactive DRG nerve cell profiles and dorsal horn fibers were reduced on the ipsilateral side at all time points. Thus, IAPP and CGRP expression are down-regulated upon axotomy. Nerve repair maintains or restores IAPP and CGRP expression in individual neurons but does not prevent the loss of CGRP/IAPP phenotype of some of these neurons in response to axotomy.     

Somatotopic redistribution of c-fos expressing neurons in the superficial dorsal horn after peripheral nerve injury

The functional somatotopic reorganization of the lumbar spinal cord dorsal horn after nerve injury was studied in the rat by mapping the stimulus-evoked distribution of neurons expressing proto-oncogene c-fos. In three different nerve injury paradigms, the saphenous nerve was electrically stimulated at C-fibre strength at survival times ranging from 40 h to more than six months: 1) Saphenous nerve stimulation from three weeks onwards after ipsilateral sciatic nerve transection resulted in an increase in the number of Fos-immunoreactive neurons within the dorsal horn saphenous territory in laminae I-II, and an expansion of the saphenous territory into the denervated sciatic territory until 14 weeks postinjury. 2) Saphenous nerve stimulation from five days onwards after ipsilateral sciatic nerve section combined with saphenous nerve crush resulted in an increase in the number of Fos-immunoreactive neurons within the dorsal horn saphenous nerve territory, and an expansion of the saphenous nerve territory into the denervated sciatic nerve territory. 3) Stimulation of the crushed nerve (without previous adjacent nerve section) at five days, but not at eight months resulted in a temporary increase in the number of Fos-immunoreactive neurons within the territory of the injured nerve, and no change in area at either survival time. The results indicate that nerve injury results in an increased capacity of afferents in an adjacent uninjured, or regenerating nerve, to excite neurons both in its own and in the territory of the permanently injured nerve in the dorsal horn. The onset and duration of the increased postsynaptic excitability and expansion depends on the types of nerve injuries involved. These findings indicate the complexity of the central changes that follows in nerve injuries that contain a mixture of uninjured, regenerating and permanently destroyed afferents.     

A motor neuron-specific epitope and the low-affinity nerve growth factor receptor display reciprocal patterns of expression during development, axotomy, and regeneration

Somatic motor neurons begin to express the transmitter synthesizing enzyme, choline acetyltransferase (ChAT) and the low-affinity nerve growth factor receptor (NGFR) during embryonic development. However, as motor neurons mature in postnatal life, they lose immunoreactivity for NGFR and acquire a motor neuron-specific epitope that is recognized by the monoclonal antibody, MO-1. The present study was undertaken to examine the effect of nerve injury in adult rats on these three developmentally regulated markers in two populations of somatic motor neurons. Unilateral transection, ligation, or crushing of the sciatic nerve resulted in a loss of MO-1 binding and a concomitant rise in immunoreactivity for NGFR within axotomized motor neurons in lumbar levels of the spinal cord. These changes, detectable within 5 days following nerve injury, are reversed with reinnervation, but persist if reinnervation is prevented by chronic axotomy. Thus, regulation of the expression of NGFR and the MO-1 epitope appears to be critically dependent upon interactions between motor neurons and target muscles. These observations are also consistent with the idea that during regeneration, neurons may revert to a developmentally immature state; in motor neurons, this state is characterized by the presence of NGFRs and the absence of the MO-1 epitope. Transection of the hypoglossal nerve, a purely motor nerve, resulted in a similar loss of MO-1 binding and a selective rise in NGFR immunoreactivity in neurons within the ipsilateral hypoglossal motor nucleus. In addition, immunoreactivity for ChAT was also lost in axotomized hypoglossal motor neurons. In contrast, injury to the sciatic nerve, which bears both sensory and motor axons, did not result in any detectable change in ChAT immunoreactivity in spinal motor neurons.     

gp130 cytokines, leukemia inhibitory factor and interleukin-6, induce neuropeptide expression in intact adult rat sensory neurons in vivo: time-course, specificity and comparison with sciatic nerve axotomy

The gp130 cytokines leukemia inhibitory factor and interleukin-6 are neuroactive cytokines associated with peripheral nerve injury. Here we show that exogenous administration of these factors selectively regulates neuropeptide phenotype in intact sensory neurons in a manner consistent with their role as injury-induced factors. Intraneural injection of leukemia inhibitory factor into the intact sciatic nerve of adult rats induces a significant increase in the percentage of neuronal profiles immunoreactive for galanin in the L4 and L5 dorsal root ganglia without altering the percentage profiles immunoreactive for vasoactive intestinal polypeptide or neuropeptide Y. Galanin-immunoreactivity was predominantly confined to those neurons which retrogradely transported and accumulated leukemia inhibitory factor. The up-regulation of galanin-immunoreactivity observed in L4 and L5 dorsal root ganglia following unilateral axotomy of the sciatic nerve was significantly reduced following continuous treatment for two weeks with a monoclonal antibody against the gp130 receptor motif. Intraneural injection of interleukin-6 into the intact sciatic nerve also significantly increased the percentage of neuronal profiles which displayed galanin-immunoreactivity but not vasoactive intestinal polypeptide or neuropeptide Y-immunoreactivity. Our results indicate that cytokines which interact with the gp130 receptor at the site of peripheral nerve injury contribute to the cell body response to axotomy. Changes in the levels of such cytokines however are insufficient to account for the complete repertoire of neuropeptide phenotypic changes associated with peripheral nerve injury.     

Distribution of the tetrodotoxin-resistant sodium channel PN3 in rat sensory neurons in normal and neuropathic conditions

The novel sodium channel PN3/alpha-SNS, which was cloned from a rat dorsal root ganglion (DRG) cDNA library, is expressed predominantly in small sensory neurons and may contribute to the tetrodotoxin-resistant (TTXR) sodium current that is believed to be associated with central sensitization in chronic neuropathic pain states. To assess further the role of PN3, we have used electrophysiological, in situ hybridization and immunohistochemical methods to monitor changes in TTXR sodium current and the distribution of PN3 in normal and peripheral nerve-injured rats. (1) Whole-cell patch-clamp recordings showed that there were no significant changes in the TTXR and TTX-sensitive sodium current densities of small DRG neurons after chronic constriction injury (CCI) of the sciatic nerve. (2) Additionally, in situ hybridization showed that there was no change in the expression of PN3 mRNA in the DRG up to 14 d after CCI. PN3 mRNA was not detected in sections of brain and spinal cord taken from either normal or nerve-injured rats. (3) In contrast, immunohistochemical studies showed that major changes in the subcellular distribution of PN3 protein were caused by either CCI or complete transection of the sciatic nerve. The intensity of PN3 immunolabeling decreased in small DRG neurons and increased in sciatic nerve axons at the site of injury. The alteration in immunolabeling was attributed to translocation of presynthesized, intracellularly located PN3 protein from neuronal somata to peripheral axons, with subsequent accumulation at the site of injury. The specific subcellular redistribution of PN3 after peripheral nerve injury may be an important factor in establishing peripheral nerve hyperexcitability and resultant neuropathic pain.     

Diabetes and axotomy-induced deficits in retrograde axonal transport of nerve growth factor correlate with decreased levels of p75LNTR protein in lumbar dorsal root ganglia

The effect of sensory neurone axotomy on the level of retrograde axonal transport of nerve growth factor (NGF) was studied in the sciatic nerve of age-matched normal and 8-week streptozocin-diabetic rats. In normal rats a 10-day sciatic nerve crush induced a 41% decrease in transported NGF, however, axotomy of sensory neurones of diabetic rats did not significantly effect the already deficient levels of NGF undergoing retrograde transport. At first sight, this result indicated that transported NGF levels in the sciatic nerve of diabetic rats are at a residual level due to deficient availability of target-derived NGF. To confirm this, the relationship of the transported NGF to the level of sensory neurone expression of the NGF receptor proteins was analysed. Western blots of L4 and L5 dorsal root ganglia (DRG) homogenates revealed no effect of axotomy and/or diabetes on the levels of the 145-kDa tyrosine kinase form of trkA. However, the expression of p75LNTR protein in the intact DRG was reduced in diabetic compared with normal rats (56%; P < 0.01), and axotomy reduced the levels in the ipsilateral ganglia of normal but not diabetic rats - as seen for NGF axonal transport. Reductions in retrograde axonal transport of NGF in both diabetes and/or axotomy were associated with the levels of p75LNTR within the lumbar DRG.     

The expression of P2X3 purinoreceptors in sensory neurons: effects of axotomy and glial-derived neurotrophic factor

We have studied the distribution and regulation of the P2X3 receptor (a ligand-gated ion channel activated by ATP) in adult dorsal root ganglion (DRG) neurons using a polyclonal antibody. P2X3 receptor immunoreactivity was normally present in about 35% of L4/5 DRG neurons, virtually all small in diameter. In the dorsal horn, P2X3 receptor expression was restricted to the terminals of sensory neurons terminating in lamina IIinner. P2X3 receptors were expressed in approximately equal numbers of sensory neurons projecting to skin and viscera but in very few of those innervating skeletal muscle. P2X3 receptors were found mostly in sensory neurons that bind the lectin IB4. After sciatic nerve axotomy, P2X3 receptor expression dropped by more than 50% in L4/5 DRG. Glial cell line-derived neurotrophic factor (GDNF), delivered intrathecally, completely reversed axotomy-induced down-regulation of the P2X3 receptor. We conclude that P2X3 receptors are normally expressed in nociceptive primary sensory neurons, predominantly the nonpeptidergic nociceptors. P2X3 receptors are down-regulated following peripheral nerve injury and their expression can be regulated by GDNF.     

ADP-ribosylation of serum proteins: evaluation as a potential tumor marker

The low-affinity p75 receptor for nerve growth factor (p75NGFR) has been implicated in mediating neuronal cell death in vitro. A recent in vitro study from our laboratory showed that the death of sensory neurons can be prevented by reducing the levels of p75NGFR with antisense oligonucleotides. To determine if p75NGFR also functions as a death signal in vivo, we have attempted to reduce its expression in peripheral sensory neurons by applying antisense oligonucleotides to the proximal end of the transected sciatic or median and ulnar nerves. We report here that antisense oligonucleotides, when applied to the proximal stump of a transected peripheral nerve, are retrogradely transported and effectively reduce p75NGFR protein levels in sensory neurons located in the dorsal root ganglia. Furthermore, treatment of the proximal nerve stump with antisense p75NGFR oligonucleotides significantly reduced the loss of these axotomized sensory neurons. These findings further support the view that p75NGFR is a death signaling molecule and that it signals death in axotomized neurons in the neonatal sensory nervous system.     

Rapid activation of post-hepatectomy factor/nuclear factor kappa B in hepatocytes, a primary response in the regenerating liver

The liver represents one of the few organs in the intact animal that has the capacity to regenerate following injury or partial hepatectomy. One of the earliest responses that has been detected in the remnant liver is the activation of post-hepatectomy factor(s) (PHF), a kappa B site DNA binding activity. We reasoned that understanding the molecular nature of PHF might provide insight into what triggers liver regeneration. We found that PHF is rapidly activated and turned over in the regenerating liver, demonstrating peak activity at 30 min post-hepatectomy and virtual disappearance by 1 h. As determined by supershift, cross-linking, and cross-linking/immunoprecipitation analyses, PHF contains intact p50/p65nuclear factor kappa B (NF-kappa B) subunits. To explore the basis for activation of PHF/NF-kappa B in the regenerating liver, we determined the level of individual Rel family subunits in the nuclei of normal and regenerating liver cells. We found evidence for nuclear translocation of p65/RelA, but other Rel family proteins including p50/NF-kappa B1 and p52/NF-kappa B2 are present at a low level in the nuclei of cells at a constitutive level pre- and post-hepatectomy and appear not to form DNA binding homodimers. The level of I kappa B-alpha falls slightly then increases at 3 h post-hepatectomy in concert with the induction of its mRNA. As demonstrated by the induction of I kappa B-alpha mRNA in hepatocytes in situ and identification of PHF/NF-kappa B in cultured hepatocytes, PHF/NF-kappa B is localized primarily in hepatocytes in the regenerating liver. This represents one of the few examples of NF-kappa B activation in the intact animal in a non-hematopoietic cell type. The activation of PHF/NF-kappa B suggests a mechanism by which hepatocytes regulate their mitogenic program during liver regeneration.     

Pituitary adenylate cyclase activating peptide expression in the rat dorsal root ganglia: up-regulation after peripheral nerve injury

Pituitary adenylate cyclase activating peptide (PACAP) is expressed in a population of capsaicin-sensitive primary sensory neurons of small to medium size in the rat. In the present report we have examined the effect of sciatic nerve injury (unilateral transection) on PACAP expression (immunocytochemistry, radioimmunoassay, in situ hybridization and northern blot analysis) in dorsal root ganglia at the lumbar level and on immunoreactive PACAP in the spinal cord and in the sciatic nerve stump. For comparison, calcitonin gene-related peptide was examined. In dorsal root ganglia of the intact side immunoreactive PACAP and PACAP messenger RNA were localised to a population of nerve cell bodies of small to medium size. In dorsal root ganglia on the injured side, PACAP-immunoreactive nerve cell bodies were more numerous and PACAP messenger RNA was considerably more abundant as studied 14 days after sciatic nerve transection. By contrast, calcitonin gene-related peptide-containing nerve cell bodies were numerous and rich in calcitonin gene-related peptide messenger RNA in dorsal root ganglia on the intact side, while after transection both the number of immunoreactive nerve cell bodies and their content of messenger RNA were markedly reduced. There were indications of axotomy-induced expression of PACAP messenger RNA in larger neurons. In the dorsal horn of the spinal cord on the intact side PACAP and calcitonin gene-related peptide-immunoreactive fibres were densely accumulated in the superficial layers. On the transected side the densities of both PACAP and calcitonin gene-related peptide-immunoreactive nerve fibres were reduced in the medial part. The data obtained indicate a marked up-regulation of PACAP in sensory neurons following peripheral nerve injury. Since PACAP depresses a C-fibre evoked flexion reflex, this may have implications for sensory transmission. Further, in view of the known promoting effects of PACAP on neuronal survival and differentiation and non-neuronal cell growth as well as its proinflammatory effects a role of PACAP in the neuronal and periaxonal tissue restoration after injury is not inconceivable.     

Overexpression of glucagon-like peptide-1 receptor in an insulin-secreting cell line enhances glucose responsiveness

Pituitary adenylate cyclase-activating polypeptide (PACAP), a member of the VIP (vasoactive intestinal polypeptide) family of peptides, has been demonstrated in neurons of the sensory system. PACAP expression of these neurons is sensitive to nerve damages such as nerve crush or axotomy. In the present study, PACAP expression in the mesencephalic trigeminal nucleus of the rat was examined after transsection of the main trunk of the masseteric nerve. The primary sensory neurons of the nucleus are considered to have purely proprioceptive functions. By quantitative in situ hybridization using a PACAP [35S]cRNA probe, an increase in PACAP mRNA was observed on the side ipsilateral to transsection already after 3 h and the expression reached a peak 24 h after surgery after which the levels gradually decreased during the next 14 days. A low and constant expression of PACAP mRNA could be seen on the side contralateral to transsection. PACAP immunoreactivity was demonstrated on the ipsilateral side after 18 h, using a specific monoclonal PACAP antibody. Co-existence of PACAP with NPY and galanin was demonstrated 7 days after transsection. Analysis of the masseteric nerve by radioimmunoassay on transsected and normal nerve stumps revealed an increase of PACAP-38 immunoreactivity in the nerve proximal to the transsection compared to the normal side (15.3 vs. 6.1 pmol/g wt). The results suggest that PACAP has a role in the early phase of adaptation to nerve injury in the proprioceptive neurons.