Keith C. Kajander

Spontaneous discharge originates in the dorsal root ganglion at the onset of a painful peripheral neuropathy in the rat.

The activity of myelinated primary afferents was recorded from the dorsal roots 1-3 days after creation of a painful peripheral neuropathy in rats. The effects on spontaneous discharge of acute transections at various points along the injured sciatic nerve and the dorsal root were determined, as were the effects of K+ channel blockers applied topically to two putative sites of impulse origin: the injured region of the nerve and the dorsal root ganglion (DRG). Transections just proximal to the nerve injury and just distal to the DRG failed to halt the discharge, but spontaneous discharge disappeared when the transection was made just proximal to the DRG (i.e. between the DRG and recording electrode). K+ channel blockers (4-aminopyridine and gallamine triethiodide) applied to the DRG increased the frequency of spontaneous discharge or initiated activity from silent fibers. Applications of K+ channel blockers to the injured region of the nerve were without effect. Thus, the spontaneous discharge and the sensitivity to K+ channel blockade seen in A beta and A delta primary afferents at the time of the onset of the neuropathic pain syndrome appear to originate in the DRG.

Transsynaptic degeneration in the superficial dorsal horn after sciatic nerve injury : effects of a chronic constriction injury, transection, and strychnine

&NA; The lumbar and cervical spinal dorsal horns of adult rats with a chronic (8 days) constriction injury of the sciatic nerve on one side (and a sham operation on the other) were examined for signs of transsynaptic degeneration. The incidence of neurons with signs of degeneration (pyknosis and hyperchromatosis; ‘dark neurons’) was significantly increased in the lumbar dorsal horn on both sides. The ipsilateral lumbar increase was significantly greater than the contralateral increase. There was no increase in the incidence of dark neurons in the cervical dorsal horns of the same rats. The distribution of lumbar dark neurons was similar bilaterally. The majority of the dark neurons were found in the sciatic nerves territory in laminae I–II. A second group of rats received the same surgery but in addition received a series of 7 daily subconvulsive doses of strychnine. Dark neurons were again found bilaterally (with ipsilateral predominance) in the sciatic nerves territory in lumbar laminae I–II, but the incidence was significantly greater than that found in the group that did not receive strychnine. The same result was obtained in a third group of strychnine‐treated rats when the sham operation was omitted. Thus the appearance of contralateral dark neurons is not dependent on unintentional nerve damage created by the sham procedure. An additional group of rats was sacrificed 8 days after receiving a unilateral sciatic nerve transection, a contralateral sham operation, and the 7 daily strychnine injections. There was no increase in the incidence of dark neurons in any of these rats. The chronic constriction injury produces signs of neuropathic pain, including hyperalgesia, allodynia, and spontaneous pain (or dysesthesia). The finding that the constriction injury evokes transsynaptic degeneration in spinal dorsal horn neurons suggests that a central anatomical abnormality might be responsible for one or more of the abnormalities of pain sensation.

Increased neuropeptide Y (NPY)-like immunoreactivity in rat sensory neurons following peripheral axotomy

The effects of peripheral axotomy (sciatic nerve transection) on the presence and distribution of neuropeptide Y (NPY) in rat dorsal root ganglion (DRG) and spinal grey matter were examined using immunocytochemistry. In normal rats and on the sham-operated side of experimental rats, NPY-like immunoreactivity (NPYir) was observed in all laminae of the lumbar spinal cord, with an especially dense concentration of immunostained axons and axonal varicosities in laminae I-II of the dorsal horn. There was no detectable NPYir in L4-L5 DRG cells from normal rats or from the sham-operated side of experimental rats. At 14 days after axotomy, there was a large ipsilateral increase in the density of NPYir axons and varicosities in the lumbar spinal cord on the side of the nerve injury; this was especially apparent in laminae III-V. In the same rats, NPYir was observed in many small, medium, and large neurons in the L4-L5 DRGs on the side of the severed nerve.

Dynorphin increases in the dorsal spinal cord in rats with a painful peripheral neuropathy

It is known that painful tissue injury evokes an increase in dynorphin in spinal neurons. It is not known, however, whether dynorphinergic systems respond similarly to the pain that accompanies peripheral neuropathy. Radioimmunoassays and immunocytochemistry were used to evaluate changes in dynorphin A(1-8) in the spinal cord of rats with a painful peripheral neuropathy. The neuropathy is the result of a constriction injury that is created by tying loose ligatures around the common sciatic nerve. Signs of abnormal pain sensations, hyperalgesia, allodynia (pain after normally innocuous stimuli), and spontaneous pain (or dysesthesia), are first detected 2-5 days after injury, reach peak severity in about 10 days, and persist for 2-3 months (Bennett, G. J.; Xie, Y.-K. Pain 33:87-107; 1988). Dynorphin increased by 5 days in cells in laminae I-II and V-VII in the lumbar spinal cord ipsilateral to the injury. This increase, maximal at 10 days (262%), was still present 20 days after the injury but was now seen only in neurons in the deep laminae (V-VII). Thus, the spinal dynorphinergic system appears to respond to neuropathic pain. Furthermore, our results suggest that dynorphinergic cells in the superficial and deep laminae may have different roles in nociception.

Effects of peripheral nerve injuries and tissue inflammation on the levels of neuropeptide Y-like immunoreactivity in rat primary afferent neurons

Changes in neuropeptide Y-like immunoreactivity (NPYir) in the rat L4 and L5 spinal cord and dorsal root ganglia (DRG) were examined after different sciatic nerve injuries (transection, loose ligation, and crush) and a localized, painful inflammation of the hind paw. Inflammation had no effect on NPYir. All the nerve injuries produced comparable increases in NPYir in ipsilateral laminae III-V axons and varicosities, and induction of NPYir in many DRG cells. Most NPYir DRG cells were medium to large (mean diameters: 40-45 microns); less than 2% of the cells had diameters of 25 microns or less. We conclude that the nerve injury-evoked increase in NPYir occurs mostly in the somata and intraspinal arbors of low-threshold mechanoreceptors; very few, if any, C-fiber afferents are involved. Nerve injury, rather than a painful condition, appears to be the stimulus for the induction of NPYir synthesis.

Up-regulation of opioid gene expression in spinal cord evoked by experimental nerve injuries and inflammation.

Opioid systems modulate nociceptive input at several levels of the CNS. At the spinal cord level neurons are present that express the genes coding for the precursors of the dynorphin and enkephalin opioid peptide families. We found that two conditions in rats, a chronic constriction injury to the sciatic nerve and peripheral inflammation, have a common consequence centrally: they evoke a large, rapid and sustained up-regulation of preprodynorphin mRNA. Both are also characterized by signs of hyperalgesia and increased primary afferent input. In contrast, there is little or no up-regulation of preprodynorphin mRNA following complete transection of the sciatic nerve or sciatic nerve crush. Furthermore, only minor alterations in the levels of preproenkephalin mRNA occur in any of the conditions, except for inflammation where the elevation is relatively small compared to that of preprodynorphin mRNA. These data imply that specific regulatory processes that include stimulation of opioid gene expression are strongly engaged in the spinal cord in certain types of peripheral nerve injuries and inflammation, but not in others. Marked and sustained up-regulation of the spinal cord dynorphin system distinguishes the chronic constriction injury model from other nerve injury models of pain.

An experimental painful peripheral neuropathy due to nerve constriction: I. Axonal pathology in the sciatic nerve

A constriction injury to the sciatic nerve of the rat produces a painful peripheral neuropathy that is similar to the conditions seen in man. The pathology of the sciatic nerve in these animals was examined at 10 days postinjury, when the abnormal pain sensations are near maximal severity. The nerves were examined with (1) complete series of silver-stained longitudinal sections of pieces of the nerve (3 cm or more) that contained the constriction injury in the center, (2) toluidine blue-stained semithin sections taken at least 1 cm proximal and 1 cm distal to the constriction, and (3) EM sections taken adjacent to those stained with toluidine blue. One centimeter or more proximal to the constriction, both myelinated and unmyelinated axons were all normal. Nearer to the constriction, extensive degeneration of myelinated axons became increasingly common, as did signs of endoneurial edema. Distal to the constriction, the nerve was uniformly edematous and full of myelinic degeneration. There was a profound loss of large myelinated axons and a distinctly less severe loss of small myelinated and unmyelinated axons. These observations show that at 10 days postinjury the constriction produces a partial and differential deafferentation of the sciatic nerves territory. The absence of degeneration in the nerve 1 cm proximal to the constriction indicates the survival of the primary afferent neurons whose axons are interrupted.

Neurochemical and Anatomical Changes in the Dorsal Horn of Rats with an Experimental Painful Peripheral Neuropathy

Many of the neuropathic pain states that occur when human somatosensory nerves are damaged by disease or trauma are thought to involve pathological changes within the spinal segments innervated by the affected nerve. Investigations in animals that have had a peripheral nerve transected or crushed have revealed several neurochemical changes within the spinal cord that may be of pathophysiological significance. Both transection and crush injuries cause a complete peripheral denervation, but their behavioral consequences differ (Wall >et al, 1979a). Rats with a transection have a marked tendency to attack the denervated region (autotomy); animals with a crush injury display autotomy infrequently and when present it is mild. It has been hypothesized that autotomy is an animal’s response to a neuropathic pain state (Wall >et al, 1979a,b; Rodin and Kruger, 1984; Coderre >et al, 1986).

Bilateral and differential changes in spinal mu, delta and kappa opioid binding in rats with a painful, unilateral neuropathy.

&NA; Quantitative receptor autoradiography was used to assess mu, delta and kappa opioid binding sites in the lumbar spinal cord of rats with neuropathic pain due to a unilateral chronic constriction injury (CCI) of the sciatic nerve. Sections from spinal segment L4 were obtained from animals of treatment groups (left side CCI, right side sham‐operated) at 2, 5 and 10 days post surgery and from control animals (left side sham‐operated, right side untreated) 10 days post surgery. Autoradiograms were made of the equilibrium binding of the highly selective opioid radioligands, 3H‐sufentanil (mu ligand), 3H‐[d‐Pen2,5]‐enkephalin (DPDPE, delta ligand) and 3H‐U69593 (Upjohn compound, kappa ligand). Computerized grain counting was performed on discrete regions of the autoradiograms corresponding to areas within laminae I–II, V and X on both sides of the spinal cord; the sciatic nerves small diameter axons terminate in these areas. With a single exception, there were no changes in binding for any of the ligands in any of the areas at 10 days post surgery in the control animals. The exception was a small increase in kappa binding in laminae I–II on the sham‐operated side. After nerve injury, however, there were marked changes (compared to the sham‐operated side of the control animals) in the amount of binding for all ligands, and most of these changes were bilateral. Mu binding was significantly increased 2–5 days post injury, bilateral to the injury in laminae V and X but only ipsilateral in laminae I–II. Mu binding in all laminae gradually declined towards control values. By day 10 significant differences remained only in lamina X. Delta binding displayed little change at 2 days post injury but declined gradually thereafter. By day 10 post injury, delta binding was significantly decreased in all three areas; these decreases were bilateral in all areas and approximately equal in laminae V and X but were significantly greater on the nerve‐injured side in laminae I–II. Kappa binding displayed a complex pattern of changes at day 2 post injury: a significant increase in ipsilateral laminae I–II and a significant increase in contralateral lamina X but no change on either side in lamina V. There was a rapid decrease in kappa binding in all three areas on both sides of the spinal cord by day 5 post injury, and these decreases were little changed by day 10. At day 5 post injury, these bilateral decreases were approximately equal in all three areas, but at day 10 the decrease in lamina X was significantly greater on the nerve‐injured side. The effects on opioid binding may be due to alterations in synaptic activity evoked by spontaneous discharges in primary afferents from the injured nerve, to activity in intraspinal circuitry, or to a pain‐ or stress‐evoked activation of descending pathways. Our observations suggest that rats, and perhaps people, with painful peripheral neuropathies may have altered responses to opiate analgesics, especially for opiates given intrathecally.

Abnormal skin temperature and abnormal sympathetic vasomotor innervation in an experimental painful peripheral neuropathy

&NA; A chronic constriction injury to the sciatic nerve of the rat produces a neuropathic pain syndrome that has many of the symptoms that are seen in humans with painful peripheral neuropathy. In particular, both the clinical and experimental conditions are accompanied by an abnormality of cutaneous temperature regulation in the painful area. A time course study was made of this phenomenon in the experimental model. In normal rats, there is little or no difference between the temperature of the two hind paws (plantar skin). After nerve injury, however, approximately 75% of the rats (N = 30) had abnormally large (greater than ± 0.9°C) temperature differences (&Dgr;T) between the affected and sham‐operated sides. The abnormal &Dgr;Ts could be either positive or negative i.e., the affected side could be hotter or colder than normal. For individual cases, the temperature abnormality was highly variable over time periods of hours to days; abnormally hot skin could switch to being abnormally cold, and vice versa, and small &Dgr;Ts in the normal range could switch between abnormal extremes. Despite this individual variability, the average &Dgr;T of the group as a whole displayed a clear evolution over the course of the 30‐day observation period: abnormally hot initially and progressing to abnormally cold. A parallel time course study was made of the status of the sympathetic vasoconstrictor innervation to the affected hind paw (plantar artery and vein). As demonstrated with a histofluorescence method that visualizes cate‐cholamines, there was a gradual loss of norepinephrine (NE)‐containing sympathetic efferents on the nerve‐injured side. The decrease was first noted on postoperative day 5 (PO5), was very marked by PO10–PO14, and progressed to a complete or nearly complete loss by PO30. There was a concommitant decrease in staining for two other substances found in vasoconstrictor efferents, dopamine‐&bgr;‐hydroxylase (DBH) and neuropeptide Y (NPY). The NE‐containing innervation of the contralateral (sham‐operated) plantar vessels appeared to be normal at all times. Lastly, in order to determine whether there was any relation between the temperature abnormality and the status of the sympathetic perivascular plexus, additional rats were sacrificed immediately after skin temperature measurement and the hind paw vessels were stained for NE. The vasculature of some abnormally cold paws had no detectable NE. Some rats that did not appear to have a temperature abnormality also had no detectable NE on the affected hind paws vasculature. The vasculature of some abnormally hot paws had normal NE. Although it is a common clinical assumption that the temperature abnormality that accompanies painful neuropathies is a reflection of the level of sympathetic vasomotor activity, the present results suggest that this is not necessarily true.