Release of immunoreactive Met-enkephalin from the spinal cord by intraventricular beta-endorphin but not morphine in anesthetized rats

Effect of beta-endorphin and morphine injected intraventricularly on the release of immunoreactive Met-enkephalin, Leu-enkephalin and dynorphin1-13 from the spinal cord was studied in anesthetized rats. Intraventricular beta-endorphin, 16 micrograms, caused a marked spinal release of immunoreactive Met-enkephalin and to a much lesser extent, of immunoreactive Leu-enkephalin while intraventricular morphine, 40 micrograms, did not cause any significant release of immunoreactive enkephalins. The release of immunoreactive Met-enkephalin was not blocked by the pretreatment with 5 mg/kg naloxone, i.p. Immunoreactive dynorphin1-13 was not released by either beta-endorphin or morphine. High performance liquid chromatographic analysis indicated that immunoreactive Met-enkephalin released by beta-endorphin had a retention time identical to [3H]Met-enkephalin. These findings in conjunction with previous pharmacological studies suggest different modes of pharmacological action for beta-endorphin and morphine.     

Preproenkephalin gene expression in the rat spinal cord after noxious stimuli

The trans-synaptically activated biosynthesis of the preproenkephalin (PPE) mRNA in the dorsal horn of the rat lumbar spinal cord was examined by an in situ hybridization histochemical technique. As a nociceptive stimulus, a small amount of formalin was injected into the right hindpaw, and quantitative and qualitative changes of PPE-mRNA expression were determined by emulsion autoradiography. Formalin injection was found to result in a significant increase in the number and signal intensity of neurons expressing PPE-mRNAs in the superficial and deep layers of the ipsilateral spinal dorsal horn. Expression of PPE-mRNA increased within 1 h after formalin injection in neurons of deep layers, but gradually for at least 24 h in neurons in the superficial layers. These results at the level of the spinal cord showed that differential responses of PPE neurons related to the pain sensation occurred trans-synaptically after nociceptive stimulation applied to the periphery.     

Leg sympathetic response to noxious skin stimuli is similar in high and low level human spinal cord injury.

OBJECTIVE: To determine if sympathetically mediated vasoconstriction in the lower extremities is injury level dependent. Although sympathetic responses have been measured in the limbs of people with high and low level SCI using blood flow measurements, including Doppler ultrasound and venous plethysmography, a direct comparison between injury levels has not been made. METHODS: Volunteers with chronic SCI were grouped according to injury level. Above T6: high level (HL, n=7), and T6 and below: low level (LL, n=6). All subjects had complete motor and sensory loss. Leg arterial flows were recorded by venous occlusion plethysmography, and continuous heart rate and mean arterial pressure (MAP) were measured. The conditioning stimulus consisted of transcutaneous stimulation to the arch of the contralateral foot. RESULTS: HL and LL subjects demonstrated a significant decrease in arterial conductance during stimulation with no significant difference found between groups. As expected, only group HL demonstrated a significant increase in MAP. CONCLUSIONS: These results support our hypothesis that local (leg) sympathetic responses are similar for both high and low level SCI. SIGNIFICANCE: While low level SCI does not typically present with autonomic dysreflexia, bouts of increased reflex sympathetic activity could have ramifications for metabolism as well as renal and motor system function.     

Systemic morphine suppresses noxious stimulus-evoked Fos protein-like immunoreactivity in the rat spinal cord.

Previous experiments have shown that noxious stimulation increases expression of the c-fos proto-oncogene in subpopulations of spinal cord neurons. c-fos expression was assessed by immunostaining for Fos, the nuclear phosphoprotein product of the c-fos gene. In this study, we examined the effect of systemic morphine on Fos-like immunoreactivity (FLI) evoked in the formalin test, a widely used model of persistent pain. Awake rats received a subcutaneous 150 microliters injection of 5% formalin into the plantar aspect of the right hindpaw. The pattern of nuclear FLI was consistent with the known nociceptive primary afferent input from the hindpaw. Dense labeling was recorded in the superficial dorsal horn (laminae I and IIo) and in the neck of the dorsal horn (laminae V and VI), areas that contain large populations of nociceptive neurons. Sparse labeling was noted in lamina IIi and in the nucleus proprius (laminae III and IV), generally considered to be nonnociceptive areas of the cord. Fos immunoreactivity was also evoked in the ventromedial gray, including laminae VII, VIII, and X. There was no labeling in lamina IX of the ventral horn. Since FLI was time dependent and distributed over several spinal segments, we focused our analysis where maximal staining was found (L3-L5) and at the earliest time point of the peak Fos immunoreactivity (2 hr). Twenty minutes prior to the formalin injection, the rats received morphine (1.0, 2.5, 5.0, or 10 mg/kg, s.c.) or saline vehicle. Two hours later, the rats were killed, their spinal cords removed, and 50 microns transverse sections of the lumbar enlargement were immunostained with a rabbit polyclonal antiserum directed against Fos. Prior treatment with morphine sulfate profoundly suppressed formalin-evoked FLI in a dose-dependent and naloxone-reversible manner. The dose-response relationship of morphine-induced suppression of FLI varied in different laminae. To quantify the effect of morphine on FLI, labeled neurons in sections taken from the L4/5 level of each rat were plotted with a camera lucida and counted. Staining in the neck of the dorsal horn (laminae V and VI) and in more ventral laminae VII, VIII, and X, was profoundly suppressed by doses of morphine which also suppress formalin-evoked behavior. Although the labeling was also significantly reduced in laminae I and II, at the highest doses of morphine there was substantial residual labeling in the superficial dorsal horn. These data indicate that analgesia from systemic opiates involves differential regulation of nociceptive processing in subpopulations of spinal nociceptive neurons.     

尿道伤害性刺激诱导的FOS阳性神经元在大鼠腰骶髓内的分布

观察了给予尿道伤害性刺激后FOS阳性神经元在大鼠腰骶髓内分布情况.在向大鼠尿道内导入100 μl 2%福尔马林后,大量FOS阳性神经元出现在脊髓腰6和骶1节段的后角内侧部和后连合核内.双侧切断阴部神经几乎可完全阻断FOS在腰骶髓的表达,但FOS表达不受切断双侧盆神经的影响.行为学观察发现神经完整的大鼠和切断盆神经的大鼠在福尔马林刺激后表现为频繁舔舐尿道外口,而切断阴部神经的大鼠则未见此明显的行为学反应.结果提示,大鼠腰6和骶1节段的后角内侧部和后连合核可能为脊髓内接受尿道伤害性信息的主要区域,尿道的伤害性信息是由阴部神经传入的.     

Inhibition of rat spinal cord dorsal horn neurons by non-segmental, noxious cutaneous stimuli

Extracellular single unit recordings were obtained from spinal cord dorsal horn neurons in halothane-anesthetized rats. Inhibitory effects induced by noxious mechanical or electrical stimuli applied to a remote area of the body surface were assessed on the spontaneous or evoked activity of these cells. Noxious mechanical stimulation inhibited 59% of the cells receiving nociceptive inputs (wide dynamic range and nociceptive specific) but only 5% of the other cell types. Inhibition produced by mechanical stimulation lasted for the full duration of stimulus application (up to 30 s) whereas inhibition produced by electrical stimulation lasted less than 500 ms. Increasing the depth of anesthesia was found to depress or abolish the inhibition.     

尿道伤害性刺激诱导的FOS阳性神经元在大鼠腰骶髓内的分布

观察了给予尿道伤害性刺激后ＦＯＳ阳性神经元在大鼠腰骶髓内分布情况。在向大尿道内导入１００μｌ２％福尔马林后,大量ＦＯＳ阳性神经元出现在脊髓要６和骶１节段的后角内侧部和后连合核内。双侧切断阴部神经几乎可完全阻断ＦＯＳ在腰骶髓的表达,但ＦＯＳ表达不受切断双侧盆神经的影响。行为学观察发现神经完整的大鼠和切断盆神经的大鼠大福尔马林刺激后表现为频繁舔舐尿道外口,而切断阴部神经的大鼠则未见此明显的行为学反应。     

The effect of chronic pain upon the response to noxious stimuli by psychiatric patients

The literature on the experimental response to noxious stimuli in patients with chronic pain has been reviewed and it has been shown that there are very few studies in this field either of “psychogenic” or “organic” pain. The results are then reported of investigations of the response to pin-prick, of thresholds obtained with the pressure algometer and of the response to electric shock measured by electronic recording of finger-movement.

The pressure algometer was the most sensitive test-instrument employed. It showed a tendency for females to react more than males and for normal subjects to react less than patients. A special group of very hypochondriacal patients reacted earliest and most. There was a tendency for anxious patients without pain to react more than depressed patients without pain, and in fact depressed patients without pain reacted relatively late. On the other hand anxious patients with pain responded late to the algometer and depressed patients with pain responded to it relatively early. Patients with hysteria and pain occupied an intermediate position between other diagnostic groups.

A test of finger-movement in response to electric shock gave clear evidence that part of the response was related to anxiety in the test situation and part to the unpleasant nature of the stimulus. It did not distinguish well between groups but did indicate that normal subjects reacted less than patients with anxiety or with pain. Whilst the algometer was the most effective indicator of differences between groups in the response to noxious stimuli there were several significant intercorrelations with other test measures. Thus the two algometer measures of P.P.T. and S.P.T. were both significantly correlated with the response to pin prick. Further, the finger-movement test showed a significant correlation with the P.P.T., so that it seems as if these tests measured a common parameter of response.

Lastly, when the responses associated with a particular site were considered it was shown that psychiatric patients without headache were inclined to react slightly less than those with headache. However, there was no significant difference between them and other psychiatric patients with pain—even with the algometer tests which involved pain on the forehead. Thus, in at least this instance, there was no significant effect of local psychogenic pain upon the response to trauma at the same site.     

Release of immunoreactive met-enkephalin from the spinal cord by intraventricular β-endorphin but not morphine in anesthetized rats

Effect of β-endorphin and morphine injected intraventricularly on the release of immunoreactive Met-enkephalin, Leu-enkephalin and dynorphin_1–13 from the spinal cord was studied in anesthetized rats. Intraventricular β-endorphin, 16 μg, caused a marked spinal release of immunoreactive Met-enkephalin and to a much lesser extent, of immunoreactive Leu-enkephalin while intraventricular morphine, 40 μg, did not cause any significant release of immunoreactive enkephalins. The release of immunoreactive Met-enkephalin was not blocked by the pretreatment with 5 mg/kg naloxone, i.p. Immunoreactive dynorphin_1–13 was not released by either β-endorphin or morphine. High performance liquid chromatographic analysis indicated that immunoreactive Met-enkephalin released by β-endorphin had a retention time identical to [³H]Met-enkephalin. These findings in conjunction with previous pharmacological studies suggest different modes of pharmacological action for β-endorphin and morphine.     

Modulation of jaw reflexes induced by noxious stimulation to the muscle in anesthetized rats

Previous studies have shown that jaw reflexes and activity patterns of the jaw muscles were modulated in the presence of jaw muscle pain. However, there is no study comparing the modulatory effects on the jaw reflexes induced by noxious stimulation to the jaw muscle. To clarify this, effects of the application of mustard oil (MO), an inflammatory irritant, into the temporalis (jaw-closing) muscle on (1) jaw-opening reflex evoked by tooth pulp stimulation (TP-evoked JOR) as a nociceptive reflex, (2) jaw-opening reflex evoked by inferior alveolar nerve stimulation as a non-nociceptive reflex and (3) jaw-closing reflex evoked by trigeminal mesencephalic nucleus stimulation as a proprioceptive reflex were investigated in anesthetized rats. The MO application induced suppression of all reflexes, and the effect on the TP-evoked JOR was more prominent than on the other reflexes. To elucidate the involvement of endogenous opioid system for the suppressive effect, a systemic administration of naloxone following the MO application was conducted. The MO-induced suppressive effect on the TP-evoked JOR was reversed by the naloxone administration. The results suggest that noxious stimulation to the jaw muscle modulate jaw reflexes particularly for the nociceptive jaw-opening reflex, and the modulatory effect includes both facilitatory and inhibitory aspects. The results also suggest that pain modulatory systems such as the endogenous opioid system play a crucial role in the suppression of the nociceptive transmissions related to nociceptive reflexes, and in some pathological states, defense reflexes may not be evoked properly.     

Recovery of spinal cord volume in postnatal rats following prenatal exposure to morphine

A previous study has shown that the spinal volume of 18-day fetal rats is decreased by 20% after maternal administration of morphine on gestation days 12-18. In the present study, the volume of the first thoracic spinal cord segment was measured in offspring of morphine or saline-injected and pairfed dams on days 6, 15 and 80 postnatally. The following volumes were measured within the segment: hemisegment, gray matter, white matter, dorsal horn, ventral horn and length. On the sixth day postnatally, the hemisegment, gray matter and dorsal horns are significantly reduced in morphine-treated and pairfed offspring. By the fifteenth day postnatally, only the gray/white ratio is reduced while the length of the segment is significantly increased in morphine-treated fetuses. The spinal measurements in pairfed offspring are normal by 15 days postnatally. These results indicate that the effect of morphine on developing spinal cord may be partially due to undernutrition; however, morphine causes a more pronounced and longer-lasting effect than undernutrition alone.     

Further studies of the effects of intranigral morphine on behavioral responses to noxious stimuli

Bilateral intranigral microinjection of morphine produces dose-related and naloxone reversible analgesic-like effects on the hot-plate and tail-flick tests. The main objectives of the present studies were to further characterize the analgesic-like effects of intranigral morphine, to determine whether these effects were related to a general impairment of sensory or motor function, and to assess their anatomical specificity. The principal findings are: (1) intranigral morphine (10 micrograms) suppresses pain-related behavior without altering responses to a variety of non-noxious auditory, visual, and somatic stimuli, and without producing motor impairment; (2) movement of injector needles approximately 1 mm rostral, dorsal, or medial to the active nigral site significantly reduces the analgesic-like effect of morphine on the tail-flick test; and (3) electrolytic lesions confined to the nigra significantly reduced the analgesic-like effect of morphine on the hot-plate test. It is concluded that the analgesic-like effects of intranigral morphine are mediated by the substantia nigra and that these effects are specifically related to pain.     

Electroacupuncture modifies the expression of c-fos in the spinal cord induced by noxious stimulation.

The present study was designed to investigate the effect of 4 Hz vs. 100 Hz electroacupuncture (EA) on c-fos expression in the spinal cord induced by noxious stimulation (NS). A second objective was to evaluate the sensitivity of these two different frequencies of EA stimulation to the opiate antagonist, naloxone. Mechanical NS was applied to the right hindpaw following 30 min of either 4 Hz or 100 Hz EA treatment and the resulting c-fos expression in the spinal cord dorsal horn was compared to that obtained in rats exposed only to the noxious stimulation. The involvement of endogenous opioids in the EA response to 4 Hz or 100 Hz stimulation frequencies was evaluated by pretreating rats with naloxone (5 mg/kg, i.p.) 10 min prior to EA. Both 4 Hz and 100 Hz EA reduced the number of c-fos-immunoreactive neurons in the spinal dorsal horn induced by noxious stimulation by 58% and 50%, respectively. The suppression of c-fos expression induced by 4 Hz EA was completely reversed by prior treatment with naloxone. On the other hand, the suppression of c-fos induced by 100 Hz EA was only partially blocked by this opiate antagonist. These data indicate that both high- and low-frequency EA are capable of inhibiting the expression of c-fos in the dorsal horn induced by NS. Low-frequency EA appears to be mediated primarily by endogenous opioid systems, while non-opioid mechanisms may be involved in mediating the analgesic effect of high frequency EA. These results support the hypothesis that EA has a direct inhibitory effect on spinal cord dorsal horn neurons and extend the results of previous studies which indicate low frequency EA is mediated by opiate sensitive circuitry, while high frequency EA is predominantly mediated by non-opioid neurotransmitters.     

The endocannabinoid system is modulated in response to spinal cord injury in rats

Endocannabinoids are lipid mediators with protective effects in many diseases of the nervous system. We have studied the modulation of the endocannabinoid system after a spinal cord contusion in rats. In early stages, lesion induced increases of anandamide and palmitoylethanolamide (PEA) levels, an upregulation of the synthesizing enzyme NAPE-phospholipase D and a downregulation of the degradative enzyme FAAH. In delayed stages, lesion induced increases in 2-arachidonoylglycerol and a strong upregulation of the synthesizing enzyme DAGL-alpha, that is expressed by neurons, astrocytes and immune infiltrates. The degradative enzyme MAGL was also moderately increased but only 7 days after the lesion. We have studied the cellular targets for the newly formed endocannabinoids using RT-PCR and immunohistochemistry against CB(1) and CB(2) receptors. We observed that CB(1) was constitutively expressed by neurons and oligodendrocytes and induced in reactive astrocytes. CB(2) receptor was strongly upregulated after lesion, and mostly expressed by immune infiltrates and astrocytes. The endocannabinoid system may represent an interesting target for new therapeutical approaches to spinal cord injury.     

Thymulin induces c-fos expression in the spinal cord of rats which is reversed by meloxicam and morphine.

Intraplantar (i.pl.) injections of thymulin have been shown to produce hyperalgesia in rats through a prostaglandin E2-dependent mechanism. This study aimed at investigating if such injections can produce sustained activation of spinal neurons by mapping the fos-like-immunoreactivity (FLI) as a marker for this activation. Our results showed that thymulin produces significant and sustained FLI in neurons located in spinal laminae known to be involved in nociception. Pretreatment with either morphine or meloxicam (a cyclooxygenase inhibitor) revealed differential effects on FLI and the hyperalgesia induced by thymulin. These findings support the hypothesis that thymulin can affect central neurons either directly or through the peripheral nerve terminals.     

Modulated expression of c-Fos in the spinal cord following noxious thermal stimulation of monoarthritic rats

Peripheral noxious stimulation evokes functional and biochemical changes in the spinal cord which results in central sensitization and hyperalgesia, but at the same time also induces the activation of inhibitory control systems. The purpose of the present study was to investigate whether the adaptive changes induced by ongoing peripheral inflammation influence the spinal cord expression of c-Fos (a commonly used marker of neuronal activity) following an additional acute noxious stimulus. Therefore, the spinal expression of c-Fos was immunohistochemically investigated following noxious thermal stimulation of a rat monoarthritic hindpaw at various time points (1, 4, 8, 21 days) after induction of monoarthritis. Compared to normal rats, c-Fos expression following ipsilateral noxious thermal stimulation of monoarthritic rats was strongly modified in the deep laminae of the dorsal horn depending on the time course of inflammation. At 1 day of monoarthritis, an enhanced ipsilateral expression (135% and 208% of normal rats in laminae III–VI and VII, respectively) and at 3 weeks a reduced expression (38% and 23% of normal rats in laminae III–VI and VII, respectively) was detected. The amount of c-Fos-positive neurons in the ipsilateral superficial laminae I and II was unchanged at all time points investigated. To assess excitability changes on the contralateral side at an early stage of inflammation, a group of monoarthritic rats received a contralateral noxious stimulus at day 1 of monoarthritis. This resulted in a potentiated expression of c-Fos ipsilateral to the acute noxious stimulus (i.e., contralateral to the monoarthritic hindpaw) restricted to lamina II (137% of normal rats) of the dorsal horn. The data showed that changes in c-Fos expression depended on the time point of noxious heat stimulation (NHS) of monoarthritic rats, and differed in the ipsi- and contralateral side of the spinal cord. In addition to a possible habituation of c-Fos expression, it may be speculated that the time course-dependent changes reflect laminae-specific modulations of excitatory and inhibitory mechanisms during monoarthritis. Further studies are necessary in order to provide more insights into the contribution of these mechanisms on noxious stimulus-evoked c-Fos expression. J. Neurosci. Res. 53:203–213, 1998. © 1998 Wiley-Liss, Inc.     

Nitric oxide mediates Fos expression in the spinal cord induced by mechanical noxious stimulation.

Immunocytochemical localization of Fos protein was used to analyze the involvement of nitric oxide (NO) in the expression of Fos in the spinal cord, induced by mechanical noxious stimulation (NS). Mechanical NS was applied to the left hindpaw 30 minutes after intrathecal administration of the NO synthase inhibitor, N omega-nitro-L-arginine methyl ester (L-NAME) and the resulting Fos expression in the spinal cord dorsal horn was compared with that obtained in rats exposed only to the mechanical NS. Pretreatment with L-NAME but not its stereoisomer N omega-nitro-D-arginine methyl ester (D-NAME), produced a dose-dependent suppression of Fos expression induced by mechanical noxious stimulation. These results indicate that NO modulates the expression of Fos in the dorsal horn induced by mechanical noxious stimulation and further support the hypothesis that NO is involved in nociceptive events occurring in the spinal cord in response to a peripheral noxious stimulus.     

Distribution of morphine in brain regions, spinal cord and serum following intravenous injection to morphine tolerant rats

In order to determine the possible contribution of altered distribution of morphine in the morphine tolerance process, the distribution of morphine was studied in brain regions and spinal cord, following its intravenous administration. Male Sprague-Dawley rats were made tolerant to morphine by implanting 6 morphine pellets, each containing 75 mg of morphine base, for 7 days. Seventy-two hours after the removal of the pellets, a time when serum morphine levels were negligible or absent and yet tolerance to the pharmacological effects of morphine was present, morphine (10 mg/kg, i.v.) was injected in placebo and morphine pellet implanted rats. At various times (5, 30, 60, 120 and 360 min) after the injection of morphine, brain regions (hypothalamus, cortex, hippocampus, midbrain, pons and medulla, striatum and amygdala), spinal cord and serum were collected. The level of morphine in the tissues was determined by using a highly sensitive and specific radioimmunoassay (RIA) method. Five minutes after morphine injection, the concentration of morphine was the highest in the hypothalamus and the lowest in amygdala. The concentration of morphine in hypothalamus, pons and medulla, hippocampus and midbrain of morphine tolerant rats was smaller than in placebo pellet implanted rats. The tissue to serum ratio of morphine in the hypothalamus, hippocampus, striatum, midbrain and cortex were also smaller in morphine tolerant than in non-tolerant rats. The concentration of morphine in brain regions with time did not exhibit linearity. At other time intervals like 30 and 60 min, the concentration of morphine in several brain regions and spinal cord was significantly higher in morphine tolerant than in non-tolerant rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

The sympathoexcitatory response following selective activation of a spinal cholinergic system in anesthetized rats

This study was performed to help elucidate the role of spinal cholinergic neurons in cardiorespiratory function by selective activation of spinal or medullary cholinergic systems in anesthetized rats. A selective site of action of cholinergic drugs on the spinal cord was obtained by refining the method of intrathecal (i.t.) drug injection to localize drug distribution to specific spinal segments. I.t. injection of the cholinesterase (ChE) inhibitor, neostigmine (NEO), produced a significant reduction in spinal, but not medullary tissue levels of ChE, and evoked marked pressor and tachycardic responses without any changes in respiratory parameters. In contrast to i.t. injection, intracisternal (i.c.) injection of NEO which inhibited both spinal and medullary ChE, produced characteristic respiratory changes--increased tidal volume and decreased respiratory rate and minute volume, as well as pressor and tachycardic responses. I.t. injection of the muscarinic antagonist, methylatropine, inhibited the cardiovascular responses to i.t. NEO, but not the cardiorespiratory responses to i.c. NEO. These cardiovascular responses to i.t. NEO were blocked by spinal transection, but not by midcollicular transection. Finally, the pressor and tachycardic responses to i.t. NEO were inhibited following peripheral alpha-adrenergic and beta-adrenergic blockade, respectively. These results indicate that activation of the spinal cholinergic system selectively produces a sympathoexcitatory response through spinal muscarinic receptor activation independent of respiratory changes. This finding is consistent with the possibility that such responses are elicited by activation of a non-cholinergic bulbo-spinal sympathoexcitatory pathway at the spinal level, or at higher centers through an ascending pathway. In either case, the spinal cholinergic system appears to be anatomically and pharmacologically distinct from the medullary pathway and may subserve a different function.