Capsaicin-sensitive vasodilatatory mechanisms in the rat substantia nigra and striatum

Summary Thermoregulatory and neurochemical effects of capsaicin microinjection into the substantia nigra (SN) or caudatus putamen (CPu) were studied in rats. Administration of capsaicin into these brain structures induced a peripheral vasodilatation which was associated with a decrease in body temperature. Pretreatment of the rats with capsaicin either as adults or neonates abolished the thermolytic response to the drug, indicating that the effect is executed specifically upon capsaicin sensitive structures. Analyses of the levels of monoamines and their metabolites in the striatum following injection of capsaicin into the SN or CPu revealed that dopaminergic neurons are not primarily involved in this effect. This view is also supported by our findings that neurochemical lesion of unilateral nigrostriatal dopaminergic neurons did not influence the vasodilatatory response. Since the pharmacological effect of intranigral capsaicin was not abolished by unilateral axotomy (hemisection) we presume a capsaicin-sensitive, non-dopaminergic descending vasodilatatory pathway from the SN.     

Ontogeny of phenylethanolamine N-methyltransferase- and tyrosine hydroxylase-like immunoreactivity in presumptive adrenaline neurones of the foetal rat central nervous system

The appearance of phenylethanolamine N-methyltransferase (PNMT)- and tyrosine hydroxylase (TH)-like immunoreactivity (LI) in the foetal rat central nervous system has been investigated. Antibodies raised against PNMT and TH were used in an indirect immunofluorescence method. Attention was focussed on areas containing putative adrenaline-containing nerve cell bodies or fibres and, using an elution-restaining technique, it was possible to analyze whether neurones contained PNMT-LI, TH-LI, or both. PNMT-immunoreactive neurones could first be visualized on day 13 of gestation, in the ventrolateral and dorsal medulla oblongata, and probably corresponding to those of the C1 and C2 groups. The number of positive cell bodies and the intensity of their fluorescent staining in these areas were not dissimilar at this stage to the number and intensity observed at 1 day postnatal, the final age studied. At day 16, PNMT-positive cells were observed for the first time in midline areas of the rostral dorsal medulla oblongata-caudal pons, associated with the medial longitudinal fasciculus. These cells probably composed the C3 cell group. Many PNMT-immunoreactive fibres could be seen at day 13 of gestation, in the medulla, and coursing both in an ascending bundle around the mesencephalic flexure and in a descending bundle toward the spinal cord. The extent of the bundles increased with gestational age, such that dense meshworks of PNMT-immunoreactive varicose fibres were visible ventral to the aqueductus Sylvii, and the periventricular and lateral regions of the hypothalamus by days 18 to 19, and in the paraventricular nucleus, the septum, and the thoracic spinal cord by day 1 after birth. A sparser fibre plexus was observed in the amygdala at day 1 postnatal. In contrast to the explosive appearance of PNMT-immunoreactive cells at day 13 of gestation, the development of TH-LI within these same neurones was much more protracted. Only rarely were TH-LI and PNMT-LI colocalized at day 13, and even at birth TH-LI could not be visualized in 5% of PNMT-immunoreactive cells in the ventrolateral medulla oblongata, and in 50% of those in the dorsal vagal complex. A similar tardy appearance of TH-LI in PNMT-immunoreactive fibres was observed also. It should be emphasized that strongly TH-immunoreactive neurones were found already at gestational day 10.5 in the medulla oblongata, caudal to the PNMT-immunoreactive cells. It is concluded that the expression of enzymes involved in the synthesis of adrenaline is not unitarily controlled, and may be partly dependent on other than epigenetic factors.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of Monoamines on Transmission from Group II Muscle Afferents in Sacral Segments in the Cat

The effects of one 5-HT_1A serotonin agonist (8-OH-DPAT) and of two α₂ noradrenaline agonists (tizanidine and B-HT 933) were tested on the transmission between group II muscle afferents and spinal neurons in the sacral segments of the spinal cord in the cat. These agonists have previously been found to depress transmission from group II muscle afferents either in the dorsal horn or in the intermediate zone of midlumbar segments, and this study addressed the question of whether their actions in the sacral segments are similarly selective. The drugs were applied ionophoretically and their effects were tested on field potentials evoked from group II muscle afferents. As judged by changes in the amplitude of the early components of these field potentials, the transmission is effectively depressed by the serotonin agonist (to 56 ± 26% after 2 min of ionophoresis of 8-OH-DPAT) but not by the noradrenaline agonists (to 97 ± 12% after 6 min of ionophoresis of B-HT 933 and to 95 ± 17% after 6 min of ionophoresis of tizanidine). These data suggest that transmission from group II muscle spindle afferents in the sacral segments is under control of serotonin releasing neurons, as in the dorsal horn of midlumbar segments, but leave open the question of the similarities or differences in the mechanisms (pre-and/or postsynaptic) of this control.     

Spinal mechanisms of te analgesic action of electroconvulsive shock

The present study investigated the spinal systems involved in the analgesic action of electroconvulsive shock (ECS). To identify such systems complete spinal transections and discrete lesions within the dorsal half of the spinal cord were performed. Complete spinal transection eliminated ECS analgesia totally, demonstrating that the observed analgesic effect is attributable to neural conduction. Lesions within the region of the dorsolateral funiculus (DLF) caused a pronounced, but incomplete, attenuation of ECS analgesia. Larger lesions of the dorsal aspects of the spinal cord including both the DLF and the dorsal column area did not result in further attenuation of analgesia. Thus, it appears that within the dorsal cord the area of the DLF contains the fibers mediating the antinociceptive action of ECS. Additional experiments were conducted to determine the neuromediators involved in ECS analgesia. Of a wide range of antagonists injected intraperitoneally (methysergide, phentolamine, haloperidol, diphenhydramine, naloxone, picrotoxin, theophylline and scopolamine), only methysergide produced a significant attenuation of ECS analgesia. In contrast, following intrathecal injections of antagonists a dose-related decrease of analgesia could be seen after the injection of methysergide, phentolamine and naloxone implicating spinal serotonin, noradrenaline and the enkephalins in the analgesic action of ECS. To assess further the interaction between the action of these neurotransmitter systems, we evaluated the effect of drug pair combinations on ECS analgesia. Intrathecal phentolamine + naloxone, methysergide + naloxone and methysergide + phentolamine were injected at doses that caused maximal attenuation of analgesia. Injections of drug combinations did not result in further attenuation of ECS analgesia. It appears that the analgesic action of ECS is mediated via systems descending within the spinal cord. The main contribution to the analgesic action of ECS is from fibers coursing within the DLF, although contribution of neural systems within the ventral spinal cord also exists. These descending analgesia systems appear to utilize serotonin, noradrenaline and endogenous opioids as neurotransmitters. Additional systems the pharmacological nature of which is still undetermined also exist.     

A re-evaluation of the neurochemical and antinociceptive effects of intrathecal capsaicin in the rat

The effect of intrathecal administration of capsaicin in the rat on thermal nociceptive thresholds and on the content of substance P, somatostatin and glutamic acid decarboxylase in the dorsal horn of the spinal cord was determined. The results suggest that the depletion of spinal cord substance P induced by capsaicin may not by itself be sufficient to explain the observed changes in noxious thermal thresholds, which may be related instead to non-specific damage to the spinal cord.     

Changes in the hypothalamic mechanisms involved in the control of body temperature induced by the early thermal environment

The experiments reported in this study were designed to investigate the influence of the early thermal environment on the functional properties of certain putative thermoregulatory neurotransmitters within the hypothalamus of the Sprague-Dawley rat. The effects on body temperature of serotonin, dopamine, carbachol, PGE₂ and endotoxin when microinjected into the anterior hypothalamic/preoptic region of the brain have been examined in warm-reared, control and warm-acclimated animals. Serotonin and PGE₂ are shown to have different effects on body temperature in warm-reared as compared to warm-acclimated and control animals. The thermoregulatory effects of intrahypothalamic dopamine are shown to be changed by the normal acclimation process, while carbachol and endotoxin have similar effects on the body temperature of all 3 groups of animals. These data suggest that the thermal environment may significantly affect the roles which specific neurotransmitters play in the control of body temperature.     

Reaction to topical capsaicin in spinal cord injury patients with and without central pain

Central neuropathic pain is a debilitating and frequent complication to spinal cord injury (SCI). Excitatory input from hyperexcitable cells around the injured grey matter zone is suggested to play a role for central neuropathic pain felt below the level of a spinal cord injury. Direct evidence for this hypothesis is difficult to obtain. Capsaicin, activating TRPV1 receptors on small sensory afferents, induces enhanced cellular activity in dorsal horn neurons and produces a central mediated area of secondary hyperalgesia. We hypothesized that sensory stimuli and capsaicin applied at and just above the level of a spinal cord injury which already is hyperexcitable, would cause enhanced responses in patients with central pain at the level of injury compared to patients without neuropathic pain and healthy controls. Touch, punctuate stimuli, cold stimuli and topical capsaicin was applied above, at, and below injury level in 10 SCI patients with central pain below a thoracic injury, in 10 SCI patients with a thoracic injury but without neuropathic pain, and in corresponding areas in 10 healthy control subjects. The study found increased responses to touch at injury level compared to controls (p = 0.033) and repetitive punctuate stimuli above and at injury level compared to controls and pain-free SCI patients (p < 0.04) but not an increased response to capsaicin in patients with central pain. These results suggest that SCI patients with below-level pain have increased responses to some but not all sensory input at the level of injury.     

Distinct lateral and medial projections of the spinohypothalamic tract of the rat

We recently described a direct nociceptive projection from the spinal cord to the hypothalamus in the rat. Several electrophysiological studies of this projection indicated that the axons of some spinohypothalamic tract neurons (SHT) reach the hypothalamus either by a lateral or by a medial route. The purpose of this study was to determine the origin of all SHT neurons that reach the hypothalamus through the lateral and the medial projections, and to investigate the possibility of ablating the SHT without damaging other important sensory and motor tracts by combining retrograde tracing techniques with axonal ablation. As compared with control cases, significant (P < .05) reductions in the number of labeled SHT neurons were encountered, 26% in the ipsilateral spinal cord following lesions of the medial projection, 67% in the contralateral spinal cord following lesions of the lateral projection, and 94% in both contra- and ipsilateral sides following lesions of both the medial and lateral projections. Bilateral lesions of the lateral projections had no effect on the distribution of labeled neurons in the spinal cord and dorsal column nuclei following injections of Fluoro-Gold (FG) into the thalamus, and a small unilateral lesion of the lateral projection reduced the ipsilateral labeling in the motor cortex following injections of FG into the pyramidal decussation. These findings suggest that most SHT neurons ascend through the contralateral lateral projection and that less than half continue in the medial projection to the ipsilateral side. They also suggest a site that can be lesioned without affecting other ascending sensory spinal pathways. © 1996 Wiley-Liss, Inc.     

Effects of neonatal capsaicin treatment on thiamine monophosphatase (TMPase) activity in the substantia gelatinosa of the rat spinal cord

The present study investigated changes in gelatinosal TMPase activity following capsaicin treatment. The gelatinosal TMPase activity was positive around postnatal day 3, and the activity disappeared when newborn rats were treated with capsaicin on postnatal days 2 or 3. The absence of TMPase activity was still observed four months after treatment. The possibility that TMPase is associated with nociception has to be considered.     

Distribution of trigeminohypothalamic and spinohypothalamic tract neurons displaying substance P receptor-like immunoreactivity in the rat

Primary afferent neurons containing substance P (SP) are apparently implicated in the transmission of noxious information from the periphery to the central nervous system, and SP released from primary afferent neurons acts on second-order neurons with the SP receptor (SPR). In the rat, nociceptive information reached the hypothalamus not only through indirect pathways but also directly through trigeminohypothalamic and spinohypothalamic pathways. Thus, in the present study, the distribution pattern of trigeminohypothalamic and spinohypothalamic tract neurons showing SPR-like immunoreactivity (SPR-LI) was examined in the rat by a retrograde tract-tracing method combined with immunofluorescence histochemistry for SPR. A substantial number of trigeminal and spinal neurons with SPR-LI were retrogradely labeled with Fluoro-Gold (FG) injected into the hypothalamic regions. These neurons were distributed mainly in lamina I of the medullary and spinal dorsal horns, lateral spinal nucleus, regions around the central canal of the spinal cord, and the lateral aspect of the deep part of the spinal dorsal horn. A number of SPR-LI neurons in the spinal parasympathetic nucleus were labeled with FG injected into the area around the paraventricular hypothalamic nucleus. Some SPR-LI neurons in the lateral spinal nucleus and the lateral aspect of the deep part of the spinal dorsal horn were also labeled with FG injected into the septal region. On the basis of the distribution areas of SPR-LI trigeminal and spinal neurons projecting to the hypothalamic and septal regions, it is likely that these neurons are involved in the transmission of somatic and/or visceral noxious information. J. Comp. Neurol. 378:508–521, 1997. © 1997 Wiley-Liss, Inc.     

Action of capsaicin on dorsal root-evoked synaptic transmission to substantia gelatinosa neurons in adult rat spinal cord slices

An action of capsaicin was investigated on dorsal root-evoked synaptic transmission to substantia gelatinosa (SG) neurons in adult rat spinal cord slices by use of the whole-cell voltage-clamp technique. In 79% of neurons examined, superfusing capsaicin (1 μM) for 30 s depressed a C-fiber-evoked excitatory synaptic current in a manner sensitive to a capsaicin-receptor antagonist, capsazepine (10 μM). On the contrary, Aδ-fiber-evoked excitatory and inhibitory synaptic currents were unaffected by capsaicin in all of cells tested. It is concluded that capsaicin specifically acts on C-afferents, resulting in an inhibition of evoked excitatory transmission to the SG; this may contribute to, at least in part, an acute analgesic action of capsaicin.     

Reduction of [3H]muscimol binding sites in rat dorsal spinal cord after neonatal capsaicin treatment

Two-day-old rats were pretreated with 50 mg/kg of capsaicin. After 3--4 months, specific binding of [3H]muscimol and [3H]strychnine was measured in membrane preparations from dorsal spinal cord. A 20-30% decrease of the number of [3H]muscimol binding sites was observed after capsaicin treatment. In contrast, [3H]strychnine binding was unchanged. The results provide indirect evidence for a presynaptic location of GABA receptors on capsaicin-sensitive primary afferent neurons.     

The termination of primary afferents within the rat dorsal horn: Evidence for rearrangement following capsaicin treatment

The mode of termination of primary afferent fibres within the superficial dorsal horn of the spinal cord was studied with anterograde tracing methods in normal rats and in animals which had been treated with capsaicin (50 mg/kg. s.c.) shortly after birth. In normal animals following injections of horseradish peroxidase (HRP) into lumbar dorsal root ganglia L4, L5, L6, a Golgi-like filling of primary afferent fibres was seen consistently within laminae I, II, and III of the dorsal horn. Several types of lamina related arborisations were observed, depending upon the survival time after HRP injection. At the earliest survival time (1–2 days) a punctate granular labelling was found in laminae I and II inner (II_i). At 3 days survival a Golgi like labelling of primary afferent axons occurred and three tiers of arborisation were seen within laminae I, II outer (II_O), and II inner (II_i), respectively. By 7 days after injection this pattern was considerably reduced and there was coarser fibre labelling within laminae I, II_O, and III. When these tracing experiments were repeated in capsaicin-treated rats in which up to 90%of unmyelinated fibres had been destroyed, evidence for a considerable loss of some inputs and rearrangement of the remaining fibres was found. At 3–5 days survival the axonal labelling within laminae I and II_i was severely depleted yet some input, particularly to lamina II_O, was present. At 7 days survival deep fibre labelling within lamina III now extended well into lamina II. Mapping of the primary afferent input following ³H-proline injections into the dorsal root ganglion and subsequent autoradiographic processing confirmed that primary afferents terminated throughout the dorsal horn but most heavily in two bands within laminae I and II_i. These two dense layers of termination were not present in capsaicin-treated animals, suggesting that they corresponded to areas of unmyelinated primary afferent fibre input. These data, taken together with histochemical observations on the distribution of substance P and fluoride-resistant acid phosphatase (FRAP)-containing primary afferents, suggests that the earliest-labelled fibre systems (3days) were unmyelinated and replaced at 7 days by predominantly A6 myelinated fibres. It is further suggested that of the three tiers of C fibre terminations seen at 3 days, the most superficial corresponds in part to those primary afferents that can be stained for substance P within lamina I while the most ventral tier within lamina II_i are those primary afferents containing FRAP. The presence of the two temporally distinct fibre systems also suggests that laminae I and II_o, receive both C and A6 primary afferent input while lamina II_i, receives only C fibre input.     

The efferent projections from the reticular formation and the locus coeruleus studied by anterograde and retrograde axonal transport in the rat

Following injections of [3H]leucine into the formatio reticularis gigantocellularis (Rgc), reticularis pontis caudalis (Rpc), reticularis pontis oralis (Rpo), reticularis mesencephali (Rmes), or the locus coeruleus (LC) of the rat, autoradiographic study revealed prominent reticuloreticular projections from all areas and secondary projections onto cranial nerve motor nuclei from most areas within the brain stem. Common long descending projections extended the full length of the spinal cord terminating in the ventromedial ventral horn and intermediate zone and more sparsely in the base of the dorsal horn and (particularly from Rgc) the region of the motoneurons. Common long ascending projections extended into the forebrain via Forel's tegmental fascicles. A dorsal branch of fibers innervated the intralaminar and midline nuclei of the thalamus. The major fiber system continued forward through Forel's fields and ascended into the pallidum from Rpo, Rmes, and LC and into the neostriatum from Rmes and LC. Fascicles from all areas also ascended in the medial forebrain bundle through the lateral hypothalamus to the lateral preoptic area, substantia innominata, and nuclei of the diagonal band. From Rpo, Rmes, and LC, fibers continued forward to reach the cerebral cortex, where the innervation was sparse and discrete from Rpo and Rmes but moderate and ubiquitous from LC. Retrograde transport of true blue and/or nuclear yellow revealed inverse gradients along the brain stem longitudinal axis of interdigitated cells respectively projecting caudally into the spinal cord (with the greatest number of cells in Rgc, Rpc, and Rpo) and rostrally into the diencephalon (with the greatest number of cells in Rmes and LC), with very few cells projecting both to the spinal cord and the diencephalon. From the basal forebrain, a large number of reticular and LC cells were retrogradely labelled, whereas from the frontal cortex, a much smaller number of reticular cells was labelled. These results document the widespread efferent projections from the reticular formation and overlapping, yet more extensive, projections from the LC.     

Spinal cord monoaminergic system response to age and cold-acclimatization in Muscovy duckling

Summary. The effect of age and cold acclimatization on the regional distribution of monoamines in duckling spinal cord was studied. In thermoneutral controls (TN), the high dopamine (DA) to norepinephrine (NE) ratios (0.25 at 4 weeks of age and 0.15 at 6 weeks of age) suggest the presence of specific (non precursor) dopaminergic pools in cervical spinal cord. DA levels and the ratio of DA to NE were lowered by age and cold exposure in the cervical cord. In TN ducklings, serotonin (5-HT) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) showed a decline with age in the spinal cord, indicating that this system is affected with development, whereas cold exposure prevents this decrease. The contents of 5-HT (+58%), 5-HIAA (+93%) and the ratio of 5-HIAA to 5-HT (+50%) are higher in the cervical spinal cord of cold acclimated than in TN ducklings. These results indicate that central monoaminergic systems are markedly affected by age and cold exposure. Received November 11, 1999; accepted March 6, 2000     

Distribution of immunoreactive dynorphin in the central nervous system of the rat

A widespread distribution of immunoreactive dynorphin (ir-Dyn) in rat brain and spinal cord was demonstrated by means of a highly specific radioimmunoassay. The highest concentrations of ir-Dyn (greater than 399 pg/mg protein) were found in hypothalamic nuclei, i.e. the premamillary, anterior hypothalamic and dorsomedial nuclei and median eminence. Relatively high concentrations of ir-Dyn (between 320 and 399 pg/mg protein) were found in other hypothalamic nuclei such as the medial and lateral preoptic, perifornical, suprachiasmatic, ventromedial nuclei and in the medulla oblongata in the area postrema and in the nucleus of the solitary tract (commissural part). Moderate levels of ir-Dyn (between 140 and 320 pg/mg protein) were found in most diencephalic areas other than the hypothalamic nuclei and further nuclei in the medulla oblongata, in the mesencephalon, pons and spinal cord. Low to moderate levels of ir-Dyn were found in the telencephalon, with lowest levels (less than 140 pg/mg protein) found in the cerebral cortex, olfactory bulb, dorsal septal nucleus, medial amygdaloid nucleus, caudate-putamen, superior collicle, cerebellum and certain areas of the reticular formation.     

Effect of capsaicin on micturition and associated reflexes in chronic spinal rats

The role of capsaicin-sensitive bladder afferents in micturition was studied in unanesthetized chronic spinal rats. Reflex voiding in response to tactile stimulation of the perigenital region appeared 5–9 days after spinal cord injury (SCI) whereas voiding induced by bladder distension occurred 2–3 weeks after SCI. The frequency and amplitude of reflex bladder contractions recorded under isovolumetric conditions were similar in chronic spinal and urethane-anesthetized CNS-intact rats. However, cystometrograms (CMGs) performed 6–8 weeks after SCI revealed that the chronic spinal rats had larger bladder capacities (1.86 ml) than CNS-intact rats (0.48 ml) and also exhibited multiple, small-amplitude, nonvoiding bladder contractions that were not detected in CNS-intact rats. Administration of capsaicin (50 mg/kg s.c.) acutely (onset 14–40 min) suppressed reflex bladder activity induced by bladder distension or by perigenital stimulation in chronic spinal animals. However, pretreatment of chronic spinal rats with capsaicin (125 mg/kg s.c.) 4 days before the experiment did not depress voiding reflexes or change bladder capacity but did eliminate the nonvoiding contractions. Inhibition of reflex bladder contractions by mechanical stimulation of rectoanal canal or the uterine cervix-vagina was not altered by pretreatment with capsaicin. These data indicate that capsaicin-sensitive bladder afferents are not essential for the initiation of reflex micturition in chronic spinal rats. However, these afferents do contribute to hyperactivity of the bladder during the filling phase of the CMG. Thus, capsaicin-sensitive bladder afferents should be evaluated as possible targets for the pharmacological treatment of bladder hyperreflexia in patients with SCI.     

Expression of c-fos-like protein as a marker for neuronal activity following noxious stimulation in the rat

C-fos is a proto-oncogene that is expressed within some neurons following depolarization. The protein product, c-fos protein, can be identified by immunohistochemical techniques. Therefore, c-fos expression might be used as a marker for neuronal activity throughout the neuraxis following peripheral stimulation. This study has analyzed patterns of c-fos expression in both control and anesthetized animals and in anesthetized rats subjected to various forms of peripheral stimulation. Labeled cells were counted in the spinal cord, brainstem, hypothalamus, and thalamus. Little c-fos immunoreactivity was found in control animals. Prolonged inhalational anesthesia increased the number of labeled cells at several brainstem sites. Noxious stimulation of anesthetized rats induced c-fos within the neuraxis in patterns consistent with data obtained from electrophysiological studies and in additional locations for which few direct electrophysiological data are available, such as the ventrolateral medulla, the posterior hypothalamic nucleus, and the reuniens and paraventricular thalamic nuclei. Gentle mechanical stimulation was ineffective in inducing c-fos-like protein. The data suggest that c-fos can be used as a transynaptic marker for neuronal activity following noxious stimulation. However, c-fos is expressed only in some kinds of neurons following peripheral stimulation, and it therefore may be an incomplete marker for nociresponsive activity. In addition, at least a few neurons express c-fos protein in the absence of noxious stimulation. Experiments analyzing c-fos expression must be designed with care, as both extraneous stimuli and anesthetic depth influence the results.     

Damage to bulbospinal serotonin-, tyrosine hydroxylase-, and TRH-containing axons occurs early in the development of experimental allergic encephalomyelitis in rats.

Spinal cord monoaminergic and peptidergic axonal damage occurring during the development of experimental allergic encephalomyelitis (EAE) was assessed using immunohistochemistry. Spinal cord axons immunoreactive for serotonin, catecholamines, or a thyrotropin-releasing hormone marker peptide were found to be markedly swollen and distorted by the earliest stage of detectable paralysis during EAE development (the flaccid tail stage). As clinical signs progressed to complete hindlimb paralysis, axonal damage became increasingly extensive. Axonal damage was equally pronounced whether EAE was induced by inoculation with purified myelin basic protein or with whole spinal cord homogenate, suggesting that the damage did not result from an immune attack directed against specific monoaminergic and/or peptidergic antigens present in the inoculant. However, two observations suggested that mechanical or chemical factors associated with the inflammatory foci contribute to the axonal damage: first, distorted axons were nearly always located adjacent to blood vessels or the pial surface, sites at which inflammation occurs during EAE. Second, the severity of axonal damage correlated with the severity of the inflammation. The early onset of axonal damage during development of EAE and the close correlation that was found between the severity of axonal damage and the severity of clinical signs suggested that the axonal damage may contribute to the clinical signs of the disease.