Z. Wiesenfeld-Hallin

Messenger plasticity in primary sensory neurons following axotomy and its functional implications

Following peripheral axotomy, long-lasting changes in the expression of neuropeptides and their receptors in primary sensory neurons are observed. These changes involve the downregulation of the excitatory peptides substance P and calcitonin gene-related peptide and the upregulation of the inhibitory peptides neuropeptide tyrosine and galanin, resulting in a reduction of transmission in the dorsal horn. The changes observed are thought to represent adaptive responses to limit the consequences of peripheral nerve damage to the organism as a whole and to promote survival and recovery of the individual neuron.

Increase of galanin-like immunoreactivity in rat dorsal root ganglion cells after peripheral axotomy

The L4 and L5 dorsal root ganglia were studied in untreated rats and rats subjected to unilateral transection of the sciatic nerve, using the indirect immunofluorescence technique and antibodies to the peptide galanin (GAL). In control rats only low numbers of small ganglion cells contained GAL-like immunoreactivity (LI). After axotomy a marked increase in the number and intensity of GAL-immunoreactive ganglion cell bodies was seen on the lesion side. Thus, some primary sensory neurons react to transection of their peripheral branches by expressing increased GAL levels. A similar reaction has been described by other groups for vasoactive intestinal polypeptide.

Chronic pain-related syndrome in rats after ischemic spinal cord lesion: a possible animal model for pain in patients with spinal cord injury

&NA; We examined a pain‐related syndrome, which includes mechanical allodynia and autotomy, in rats after ischemic spinal cord injury photochemically induced by laser irradiation for 5–20 min. This procedure results in an acute allodynia‐like phenomenon which lasts for several days and is possibly related to dysfunction of the GABAB system in the spinal cord. In some animals this is followed by a chronic allodynia‐like symptom with an onset varying between 1 week and 1.5 months after injury, expressed as a clearly painful reaction to light pressure applied to a skin area at or near the dermatome of the injured spinal segments. In the majority of rats the allodynia persists over several months, in some cases accompanied by autotomy of the hind paws. Pharmacological studies indicated that the allodynia in the majority of rats could be relieved by systemic tocainide (75 mg/kg). Morphine was only effective at a sedative dose (5 mg/kg). The allodynia was not relieved by baclofen, muscimol, clonidine or carbamazepine. Low‐dose systemic pentobarbital (5 mg/kg) had a slight beneficial effect. Guanethidine (20 mg/kg s.c.) did not abolish the allodynia in most of the rats. Histological examination revealed massive damage in the spinal cord. The dorsal roots of the irradiated segments were also injured. No morphological abnormalities were seen in the dorsal root ganglia. The mechanism that may account for this chronic pain‐related syndrome in spinally injured rats probably involves abnormalities in the central nervous system. The allodynia seen in chronic spinally injured rats was similar to some painful symptoms in patients after spinal cord injury or stroke. It is suggested that the chronic allodynia‐like phenomenon may represent an animal model for studying the mechanisms of chronic central pain.

Allodynia-like effects in rat after ischaemic spinal cord injury photochemically induced by laser irradiation

&NA; We report behaviours suggesting the presence of allodynia elicited by non‐noxious brushing and mechanical pressure following photochemically induced ischaemic spinal cord injury in the rat. Female rats were intravenously injected with Erythrosin B and the T10 vertebra was irradiated with a laser beam for 1, 5 or 10 min. These procedures initiated an intravascular photochemical reaction, resulting in ischaemic spinal cord injury. After irradiation a clear allodynia was observed in most rats, The animals vocalized intensely to light touch during gentle handling and were clearly agitated to light brushing of the flanks. The vocalization threshold in response to the mechanical pressure measured with von Frey hairs was markedly decreased during this period. In some animals the existence of spontaneous pain was suggested by spontaneous vocalization. The duration of the allodynia varied among animals from several hours to several days. The severity and duration of allodynia seemed not to be related to the duration of irradiation. In sham‐operated rats a slight, transient allodynia was also noted around the wound within a few hours after surgery, which was effectively relieved by systemic morphine (2 mg/kg, i.p.). Morphine (2 mg/kg, i.p.) also partially relieved the allodynia in spinally injured rats 4 h after irradiation. However, morphine, even at a higher dose (5 mg/kg, i.p.), failed to alleviate the allodynia in spinally injured rats 24–48 h after the injury. Systemic injection of the GABAB agonist baclofen (0.01‐0.1 mg/kg, i.p.), but not the GABAA agonist muscimol (1 mg/kg, i.p.), effectively relieved allodynia during this period. Pretreatment with guanethidine 24 h and just prior to the irradiation (20 mg/kg, s.c.) did not prevent the occurrence of allodynia in spinal cord injured rats, The present observation is the first to show that ischaemic spinal cord injury could result in cutaneous mechanical allodynia. This phenomenon is resistant to morphine and may not involve the sympathetic system. Histological examination of allodynic animals 3 days after spinal cord injury revealed considerable morphological damage in the dorsal spinal cord of a rat irradiated for 5 min. The related dorsal roots were also slightly affected in this animal, while the dorsal root ganglia were normal. However, in rats irradiated for 1 min, despite the existence of strong allodynia, no damage could be found at this time in the spinal cord, dorsal roots or dorsal root ganglia. It is suggested that functional deficits in the GABAB system in the spinal cord may be related to this allodynia‐like phenomenon. Allodynia following laser‐induced spinal cord injury may be a useful pain model for testing the efficacy of analgesic drugs against central pain of ischaemic origin.

The effects of intrathecal galanin and C-fiber stimulation on the flexor reflex in the rat

Galanin (GAL) was applied intrathecally (i.t.) at the lumbar level in decerebrate, spinalized, unanesthetized rats. GAL had no effect on the amplitude of the monosynaptic reflex over a wide concentration range, but at low concentrations if briefly facilitated the flexor reflex and at higher concentrations the facilitation was sometimes followed by a depression. GAL decreased the facilitatory effect of a conditioning stimulus train to C-fibers in the sural nerve. The depressive effect of GAL could be prevented by the i.t. coadministration of calcitonin gene-related peptide (CGRP), but not substance P (SP) and was not reversed by i.t. naloxone or bicuculline. The results illustrate the complex effect of GAL on the spinal cord, possibly exhibiting a biphasic effect. The observed effects on the flexor reflex are probably not due to changes in the excitability of motoneurons. Descending inhibitory pathways or local inhibitory non-GAL interneurons probably are not involved in the depressive effect of GAL. The possibility that the observed effects are related to primary sensory afferents containing not only GAL but also CGRP, and/or to local GAL neurons in the dorsal horn is discussed.

On the Role of Galanin, Substance P and Other Neuropeptides in Primary Sensory Neurons of the Rat: Studies on Spinal Reflex Excitability and Peripheral Axotomy.

The interaction of intrathecally (i.t.) applied galanin (GAL) with substance P (SP), calcitonin gene‐related peptide (CGRP), vasoactive intestinal polypeptide (VIP), somatostatin (SOM) and C‐fibre conditioning stimulation (CS) with regard to their effects on the spinal nociceptive flexor reflex was studied in decerebrate, spinalized, unanaesthetized rats with intact or sectioned sciatic nerves. SP, CGRP, VIP and SOM applied onto the surface of lumbar spinal cord or a brief CS train (1 Hz, 20 s) to the sural nerve facilitated the flexor reflex for several minutes in animals with intact or sectioned nerves. Pretreatment with GAL, which by itself had a biphasic effect on the flexor reflex in a dose‐dependent manner, antagonized the reflex facilitation induced by sural CS before and after sciatic nerve section. SP‐induced facilitation of the flexor reflex was antagonized by GAL in rats with intact sciatic nerves, but not after nerve section. In contrast, VIP‐induced reflex facilitation was antagonized by GAL only after sectioning of the sciatic nerve. GAL was effective in antagonizing the facilitatory effect of CGRP under both situations, but had no effect on SOM‐induced facilitation. A parallel immunohistochemical study revealed that after sciatic nerve section GAL‐like immunoreactivity (LI) and VIP‐LI are increased in the dorsal root ganglia and that these two peptides coexist in many cells. The present results indicate that GAL antagonizes the excitatory effect of some neuropeptides which exist in the spinal cord. This antagonism could explain the inhibitory effect of GAL on C‐fibre CS‐induced facilitation of the flexor reflex, which is presumably due to the release of some of these neuropeptides from the terminals of primary afferents. Furthermore, the interaction between GAL and other neuropeptides is altered by sciatic nerve section, paralleling changes in the levels of these neuropeptides in primary afferents and their pattern of coexistence after nerve section. It is proposed that SP and CGRP are important mediators of the spinal flexor reflex in intact rats. However, after axotomy VIP may replace SP in this capacity, paralleling the decrease in SP and marked increase in VIP levels. In general the study provides further support for involvement of peptides in sensory function.

Spinal substance p and N-methyl-d-aspartate receptors are coactivated in the induction of central sensitization of the nociceptive flexor reflex

We have studied the effects and interactions of the neurokinin-1 receptor antagonist CP-96,345 and the N-methyl-D-aspartate receptor/channel blocker MK-801, both applied intravenously, on the flexor reflex and on the facilitation of the flexor reflex by conditioning stimulation of cutaneous C-afferents in decerebrate, spinalized, unanesthetized rats. The flexor reflex was evoked by subcutaneous electrical stimuli applied to the sural nerve innervation area 1/min at an intensity that activated C-fibers and was recorded as electromyogram from the ipsilateral hamstring muscles. The magnitude of the baseline flexor reflex was usually highly stable in the course of the experiments without experimental manipulations. The same stimulus was used as a conditioning train (0.9 Hz, 20 shocks) and caused a brief facilitation of the flexor reflex, which was maximal 0.5 and 1 min after stimulation (255.1 +/- 23.6% over baseline). During the course of the conditioning stimulus train, the reflex magnitude was gradually increased (wind-up). MK-801 (0.1 and 0.5 mg/kg) consistently depressed the polysynaptic flexor reflex. At a dose of 0.5 mg/kg, but not 0.1 mg/kg, MK-801 reduced the wind-up and blocked the facilitation of the flexor reflex induced by the conditioning stimulus by 90%. The facilitatory effect of 7 pmol intrathecal substance P was also partially reduced by MK-801. CP 96,345 (1 and 3 mg/kg) did not depress the flexor reflex, but dose-dependently antagonized reflex facilitation by the conditioning stimulus train, similarly to its antagonism of intrathecally applied 7 pmol substance P-induced facilitation.(ABSTRACT TRUNCATED AT 250 WORDS)

Central and peripheral expression of galanin in response to inflammation

Using in situ hybridization, immunohistochemistry and receptor binding methodology, the galanin messenger RNA levels, galanin binding and galanin-like immunoreactivity were examined in rats injected with carrageenan into the left hindpaw. Three days after injection, a distinct increase (63%) in galanin messenger RNA-positive neurons was observed in the medial laminae I and II of the ipsilateral dorsal horn (lumbar 4 and 5) as compared to the contralateral side. However, no alteration was found in galanin binding and galanin-like immunoreactivity in the dorsal horn. In dorsal root ganglia (lumbar 5), inflammation induced a significant decrease in galanin messenger RNA (39%) and galanin peptide (47%) on the ipsilateral side. Galanin binding was not detected in dorsal root ganglia, neither on the inflammatory nor on the control side. Increased levels of galanin-like immunoreactivity and galanin messenger RNA were seen in cells in the inflamed dermis and epidermis, especially in stratum granulosum. Most of the galanin-immunoreactive cells contained ED1-like immunoreactivity, a marker for macrophages. A strong galanin binding was seen in the inflamed dermis. Such binding sites may be targets for galanin released from local cells in inflamed dermis. Taken together, our results suggest that both neuronal and non-neuronal galanin or a galanin-like peptide is involved in the response to inflammation.

Effect of Peripheral Axotomy on Expression of Neuropeptide Y Receptor mRNA in Rat Lumbar Dorsal Root Ganglia

Using in situ hybridization, the expression of the mRNA for a neuropeptide Y (NPY) receptor, was studied in lumbar (L) 4 and 5 dorsal root ganglia (DRGs) of normal rats and at various intervals after unilateral sciatic nerve transection. Twenty percent of all normal DRG neurons were NPY receptor mRNA‐positive, and the majority of these neurons were of the small type, with only a few labelled medium‐sized and large neurons. In L5 normal ganglia NPY receptor mRNA colocalized with substance P, calcitonin gene‐related peptide and galanin mRNAs in small neurons, but not in medium‐sized or large neurons containing these peptides. NPY receptor mRNA was not observed in somatostatin or nitric oxide synthase mRNA‐positive neurons. Sciatic nerve transection induced a marked decrease in NPY receptor mRNA levels. However, in parallel there was a transient increase in the number of NPY receptor mRNA‐positive small neuron profiles, but the intensity of labelling was mostly very low, although a few strongly labelled, small neuron profiles were also encountered. In addition, axotomy caused a marked increase in the number of NPY receptor mRNA‐positive large neuron profiles in the ipsilateral DRGs, and they constituted 15–20% of counted DRG neuron profiles and 45–65% of counted large neuron profiles, 7–28 days after axotomy. In L5 DRGs, ipsilateral to the axotomy, NPY receptor mRNA colocalized with NPY mRNA in many large and some medium‐sized neuron profiles, with galanin mRNA in some small, medium‐sized and large neuron profiles and with vasoactive intestinal polypeptide mRNA in some small and medium‐sized neuron profiles and a few large profiles. Occasionally, NPY receptor mRNA was observed in nitric oxide synthase mRNA‐positive small neurons. In the dorsal horn, NPY receptor mRNA‐positive small neurons were concentrated in lamina II at L4 and L5 levels, and were scattered in deeper laminae. No marked changes were observed ipsilateral to the axotomy. No NPY receptor mRNA‐positive cells were found in the normal rat gracile nucleus, or in this nucleus after axotomy. These results show that a NPY receptor may be a prejunctional receptor in primary afferent neurons and play a role in the modulation of somatosensatory information, both in normal and lesioned primary afferent DRG cells. However, axotomy induced a distinct shift in NPY receptor mRNA expression from small to large neurons, indicating that sensitivity to NPY is switched from one modality to another. Thus, not only several sensory neuropeptides, as shown in previous studies, but at least also one of the peptide receptors change their expression dramatically in response to axotomy, suggesting complex adaptive responses.

Galanin and spinal nociceptive mechanisms: recent advances and therapeutic implications

Galanin is a peptide consisting of 29 or 30 (in humans) amino acids that is present in sensory and spinal dorsal horn neurons. Endogenous galanin may have an important modulatory function on nociceptive input at the spinal level. In addition, exogenously administered galanin exerts complex effects on spinal nociceptive transmission, where inhibitory action appears to predominate. Peripheral nerve injury and inflammation, conditions associated with chronic pain, upregulate the synthesis of galanin in sensory neurons and spinal cord neurons, respectively. Hence, the sensory effect of galanin may be increased under these conditions, raising the possibility that modulation of the activity of the galanin system may produce antinociception.