Pain sensitivity following loss of cholinergic basal forebrain (CBF) neurons in the rat.

Flexion/withdrawal reflexes are attenuated by spinal, intracerebroventricular (ICV) and systemic delivery of cholinergic agonists. In contrast, some affective reactions to pain are suppressed by systemic cholinergic antagonism. Attention to aversive stimulation can be impaired, as is classical conditioning of fear and anxiety to aversive stimuli and psychological activation of stress reactions that exacerbate pain. Thus, in contrast to the suppressive effects of cholinergic agonism on reflexes, pain sensitivity and affective reactions to pain could be attenuated by reduced cerebral cholinergic activation. This possibility was evaluated in the present study, using an operant test of escape from nociceptive thermal stimulation (10 °C and 44.5 °C) before and after destruction of basal forebrain cholinergic neurons. ICV injection of 192 IgG-saporin produced widespread loss of basal forebrain cholinergic innervation of the cerebral cortex and hippocampus. Post-injection, escape from thermal stimulation was decreased with no indication of recovery for upto 19 weeks. Also, the normal hyperalgesic effect of sound stress was absent after ICV 192-sap. Effects of cerebral cholinergic denervation or stress on nociceptive licking and guarding reflexes were not consistent with the effects on operant escape, highlighting the importance of evaluating pain sensitivity of laboratory animals with an operant behavioral test. These results reveal that basal forebrain cholinergic transmission participates in the cerebral processing of pain, which may be relevant to the pain sensitivity of patients with Alzheimer's disease who have prominent degeneration of basal forebrain cholinergic neurons.

A preclinical model of hyperalgesia following spinal stenosis/compression.

BACKGROUND: Identification of mechanisms for pain/hyperalgesia following spinal cord injury requires long-term evaluation of individual subjects because of the variability in effect over time for humans. METHODS: Rats were trained on an operant escape task that determined their preference for occupancy of a brightly lit compartment versus a dark compartment with a floor preheated to 10, 32 or 44.5 °C. Following determination of baseline preferences, the animals received extradural implantation of a small piece of polymer in the thoracic spinal canal. The polymer narrowed the spinal canal and compressed the spinal cord. Post-operative tests of escape preference were conducted over 23 weeks (experiments 1 and 2) and 62 weeks (experiment 3), permitting statistical evaluation of individual effects. RESULTS: Spinal stenosis/compression produced hyperalgesia for cold and/or heat stimulation (17 animals; 77%), no post-operative change in sensitivity (4 animals) or hypoalgesia for cold or heat (2 animals). When hyperalgesia occurred, it developed gradually over 4 months. Following removal of the polymer in experiment 3, heat sensitivity returned to baseline levels for four of four animals that had been hyperalgesic when the polymer was in place, but cold hyperalgesia was retained for four of five animals. Overall, post-operative changes in cold and heat sensitivity were not strongly related, indicating that different mechanisms were responsible for enhanced sensitivity to 10 and 44.5 °C. CONCLUSIONS: Histology revealed that hyperalgesia occurred when there was: (1) damage to spinal white matter; or (2) cystic cavitation; or (3) compression and distortion of the spinal cord without an obvious loss of grey or white matter.

Sensory function after cavernous haemangioma: a case report of thermal hypersensitivity at and below an incomplete spinal cord injury.

STUDY DESIGN: Case report of a 42-year-old woman with non-evoked pain diagnosed with a cavernous C7-Th6 spinal haemangioma. OBJECTIVES: To assess the effect of intramedullary haemorrhage (IH) on nociception and neuropathic pain (NP) at and below an incomplete spinal cord injury (SCI). SETTING: Sensorimotor Function Group, Hospital Nacional de Parapléjicos de Toledo (HNPT). METHODS: T2*-susceptibility weighted image (SWI) magnetic resonance imaging (MRI) of spinal haemosiderin and a complete pain history were performed 8 months following initial dysaesthesia complaint. Thermal pain thresholds were assessed with short 1 s stimuli, while evidence for central sensitization was obtained with psychophysical electronic Visual Analogue Scale rating of tonic 10 s 3 °C and 48 °C stimuli, applied at and below the IH. Control data were obtained from 10 healthy volunteers recruited from the HNPT. RESULTS: Non-evoked pain was present within the Th6 dermatome and lower legs. T2*-SWI MRI imaging detected extensive haemosiderin-rich IH (C7-Th5/6 spinal level). Cold allodynia was detected below the IH (left L5 dermatome) with short thermal stimuli. Tonic thermal stimuli applied to the Th6, Th10 and C7 dermatomes revealed widespread heat and cold allodynia. CONCLUSION: NP was diagnosed following IH, corroborated by an increase in below-level cold pain threshold with at- and below-level cold and heat allodynia. Psychophysical evidence for at- and below-level SCI central sensitization was obtained with tonic thermal stimuli. Early detection of IH could lead to better management of specific NP symptoms, an appreciation of the role of haemorrhage as an aggravating SCI physical factor, and the identification of specific spinal pathophysiological pain mechanisms.

Effects of age on thermal sensitivity in the rat.

Age-dependent changes in thermal sensitivity were evaluated with reflex- and operant-based assessment strategies in animals ranging in age from 8 to 32 months. The impact of inflammatory injury on thermal sensitivity was also determined in animals of different ages. The results showed that operant measures of escape behavior are needed to demonstrate significant changes in thermal sensitivity across the life span of female Long-Evans rats. Increased escape from both heat (44.5 degrees C) and cold (1.5 degrees C-15 degrees C) was observed for older animals, with a greater relative increase in sensitivity to cold. Physical performance deficits were demonstrated with aging but were not associated with changes in escape responding. Reflex responding to cold stimulation was impaired in older animals but was also influenced by physical disabilities. Reflex responding to heat was not affected by increasing age. Inflammation induced by formalin injections in the dorsal hindpaw increased thermal sensitivity significantly more in older animals than in their younger counterparts.

Long-lasting hyperalgesia and sympathetic dysregulation after formalin injection into the rat hind paw.

Subcutaneous formalin injection has been used extensively to evaluate acute effects (over several hours) of chemical nociceptive stimulation on nociceptive reflexes. Also, a persistent hyperreflexia for mechanical and thermal stimulation, lasting 3 weeks after formalin injection, has been revealed and related to microglial activation in the spinal dorsal horn. The present study demonstrates more prolonged effects of formalin injection, lasting 6 weeks, on operant escape from nociceptive thermal stimulation. Operant escape requires cerebral processing of nociceptive input and can detect effects that are not limited to spinal or spinal-brain stem-spinal reflex circuits. Compared with rats injected with saline, escape responding to 44.5 degrees C and 47 degrees C stimulation was increased after bilateral s.c. injection of 5% formalin into the dorsal hind paws. The hyperalgesia outlasted visible signs of trauma (e.g. paw edema). Responses to 36 degrees C were not altered after formalin injection, providing a control for effects of the peripheral injury on activity levels or exploratory tendencies. Skin temperature recordings from the forepaws and contralateral hind paw during 44.5 degrees C stimulation of the left hind paw provided an indirect measure of cutaneous blood flow in formalin- and saline-injected animals. Normal reductions in skin temperature during thermal stimulation were attenuated (nearly eliminated) at 1 and 2 weeks after formalin injection and partially recovered by 10 weeks. Thus, formalin-induced tissue injury produced a long-term secondary hyperalgesia, accompanied by a reduced sympathetic responsivity. The similar time-course for these phenomena suggests that there are mechanistic linkages between focal injury, autonomic dysregulation and enhanced pain sensitivity.

Opioid modulation of reflex versus operant responses following stress in the rat.

In pre-clinical models intended to evaluate nociceptive processing, acute stress suppresses reflex responses to thermal stimulation, an effect previously described as stress-induced "analgesia." Suggestions that endogenous opioids mediate this effect are based on demonstrations that stress-induced hyporeflexia is enhanced by high dose morphine (>5 mg/kg) and is reversed by naloxone. However, reflexes and pain sensations can be modulated differentially. Therefore, in the present study direct comparisons were made of opioid agonist and antagonist actions, independently and in combination with acute restraint stress in Long Evans rats, on reflex lick-guard (L/G) and operant escape responses to nociceptive thermal stimulation (44.5 degrees C). A high dose of morphine (>8 mg/kg) was required to reduce reflex responding, but a moderate dose of morphine (1 mg/kg) significantly reduced escape responding. The same moderate dose (and also 5 mg/kg) of morphine significantly enhanced reflex responding. Naloxone (3 mg/kg) significantly enhanced escape responding but did not affect L/G responding. Restraint stress significantly suppressed L/G reflexes (hyporeflexia) but enhanced escape responses (hyperalgesia). Stress-induced hyperalgesia was significantly reduced by morphine and enhanced by naloxone. In contrast, stress-induced hyporeflexia was blocked by both naloxone and 1 mg/kg of morphine. Thus, stress-induced hyperalgesia was opposed by endogenous opioid release and by administration of morphine. Stress-induced hyporeflexia was dependent upon endogenous opioid release but was counteracted by a moderate dose of morphine. These data demonstrate a differential modulation of reflex and operant outcome measures by stress and by separate or combined opioid antagonism or administration of morphine.

Anti-nociceptive effects of selectively destroying substance P receptor-expressing dorsal horn neurons using [Sar9,Met(O2)11]-substance P-saporin: behavioral and anatomical analyses.

Lumbar intrathecal injections of substance P-saporin (SP-sap) destroy dorsal horn neurons that express the neurokinin-1 receptor (NK-1R) resulting in decreased responses to a range of noxious stimuli and decreased hyperalgesia and allodynia. Forebrain injections of SP-sap produce considerable non-specific damage raising some concern about use of this toxin in vivo. The more stable and selective substance P congener, [Sar9,Met(O2)11]substance P coupled to saporin (SSP-sap) produces much more selective forebrain lesions at significantly lower doses. The present study sought to determine the anatomic and nocifensive behavioral effects of lumbar intrathecal injections of the more precisely targeted SSP-sap. On the basis of loss of lamina I NK-1R staining, lumbar intrathecal SSP-sap was seven times more potent than SP-sap and produced no loss of NK-1R expressing neurons in deeper laminae (III-VI or X). Transient decreases in hotplate responding occurred at 44 degrees C and 47 degrees C but not 52 degrees C during the first 3 weeks after SSP-sap injection with return to baseline by 4 weeks. Operant escape responses were reduced at 0.3 degrees C, 44 degrees C and 47 degrees C for at least 4 months. In the formalin test, SSP-sap also was about seven times more potent than SP-sap in reducing phase two behavior in both female Long Evans and male Sprague-Dawley rats. Both SSP-sap and SP-sap reduced formalin-induced FOS expression in deep and superficial laminae of the L4 dorsal horn in parallel with the reduction in phase 2 behavior. In summary, SSP-sap is highly effective in destroying lamina I NK-1R expressing neurons, without loss of deep NK-1R neurons. The behavioral effects of SSP-sap are similar to SP-sap suggesting that the antinociceptive effects of both toxins are indeed due to selective loss of NK-1R neurons in lamina I. SSP-sap is an attractive agent for possible treatment of chronic pain.

Overall fibromyalgia pain is predicted by ratings of local pain and pain-related negative affect--possible role of peripheral tissues.

OBJECTIVES: Despite variable numbers and intensities of local pain areas, fibromyalgia (FM) patients can provide overall clinical pain ratings. We hypothesized that the overall clinical pain is largely determined by the pain intensity of local body areas. Thus, we assessed the role of local body pains as predictors of overall clinical pain in FM patients. METHODS: Ratings of overall clinical pain intensity and pain-related negative affect (PRNA) were obtained from 277 FM patients. In addition, the patients identified painful body areas by shading a body pain diagram and rated the intensity of each pain area using a mechanical visual analogue scale (VAS). Hierarchical regression analyses were used to examine predictors of overall clinical FM pain intensity including PRNA, number of local pain areas, and maximal/average intensity of local pain areas. RESULTS: The average overall clinical pain rating of all FM patients was 4.6 (S.D. 2.3) VAS. The PRNA accounted for 19%, number of painful body areas for 9% and maximal/average local pain for 27% of the variance of overall clinical FM pain (P-values < 0.001). The combination of all factors predicted 55% of the variance in overall clinical pain intensity of FM patients. CONCLUSION: Peripheral factors (maximal/average local pain and number of painful body areas) predicted most of the variance of overall clinical FM pain, suggesting that the input of pain by the peripheral tissues is clinically relevant. About 19% of the pain variance was predicted by PRNA. Thus, peripheral pain and negative affect appear to be particularly relevant for overall FM pain and may represent important targets for future therapies.

Differential effects of stress on escape and reflex responses to nociceptive thermal stimuli in the rat.

Acute stress has been shown to increase latencies of nociceptive reflexes, and this effect is considered evidence for stress-induced analgesia. However, tests for nociception that rely on motivated operant escape assess cerebral processing of pain and could be modulated independent of reflex responses. We therefore compared the effects of an acute stressor (restraint) on escape responses and lick/guard reflexes to stimulation of the paws by a thermally regulated floor. Testing sessions included a pre-test exposure to 36 degrees C, followed by a test trial in which either escape from 44 or 36 degrees C or reflex responses to 44 degrees C were observed. Behavioral responses to stress were assessed during a three day period, with baseline testing on day 1, post-stress or control testing on day 2, and evaluation of long-term stress effects on day 3. On day 2, half the animals received 15 min of restraint stress, followed by 15-min pre-test and test trials. Licking and guarding responses to thermal stimulation during 44 degrees C test trials were significantly reduced by restraint stress, confirming previously reported stress effects on nociceptive reflexes. In contrast, learned escape responses to the same thermal stimulus were significantly enhanced after stress. The increase in operant sensitivity suggests that acute restraint, a form of psychological stress, produces hyperalgesia for a level of thermal stimulation that preferentially activates C nociceptors. These results are discussed in relation to studies involving physical or psychological forms of stress, different nociceptive stimuli, and assessment strategies used to evaluate thermal pain sensitivity.

Intrathecal substance p-saporin attenuates operant escape from nociceptive thermal stimuli.

Destruction of neurons in the superficial dorsal horn that express substance P receptor (NK-1R) has been reported to block development of behavioral hypersensitivity following peripheral sensitization of nociceptors. Baseline sensitivity was not altered in these rat models that assessed innate reflex responses (i.e. hind-paw withdrawal to thermal or mechanical stimulation). In the present study, we evaluated effects of intrathecal substance P-saporin (SP-sap), a toxin selective for cells expressing NK-1R, on operant escape responses of rats to thermal stimulation. For comparison, lick/guard reflex testing was performed. Injection of a modest dose (175 ng) of SP-sap into the lumbar subarachnoid space produced a partial loss of lamina I/II NK-1R-expressing dorsal horn neurons but did not affect NK-1R-expressing neurons in deeper laminae. Lick/guard responses to 0.3, 44 or 47 degrees C were not affected after SP-sap treatment, but escape responses to these temperatures were significantly attenuated. Three hours after application of mustard oil to the dorsal surface of both hind paws, escape from 44 degrees C was enhanced for controls but not SP-sap-treated rats. Lick/guard responses were enhanced by mustard oil for both SP-sap and control animals. Administration of morphine (1.0 mg/kg, s.c.) before testing decreased escape responding at 47 degrees C for both controls and SP-sap rats. Thus, partial loss of NK-1R-expressing neurons in the superficial dorsal horn attenuated thermal nociceptive sensitivity and prevented secondary hyperalgesia when studied with an operant algesia assay, in contrast to innate reflexes which were less sensitive to modification by intrathecal SP-sap.

Feasibility and safety of neural tissue transplantation in patients with syringomyelia.

Transplantation of fetal spinal cord (FSC) tissue has demonstrated significant potential in animal models for achieving partial anatomical and functional restoration following spinal cord injury (SCI). To determine whether this strategy can eventually be translated to humans with SCI, a pilot safety and feasibility study was initiated in patients with progressive posttraumatic syringomyelia (PPTS). A total of eight patients with PPTS have been enrolled to date, and this report presents findings for the first two patients through 18 months postoperative. The study design included detailed assessments of each subject at multiple pre- and postoperative time points. Outcome data were then compared with each subject's own baseline. The surgical protocol included detethering, cyst drainage, and implantation of 6-9-week postconception human FSC tissue. Immunosuppression with cyclosporine was initiated a few days prior to surgery and continued for 6 months postoperatively. Key outcome measures included: serial magnetic resonance imaging (MRI) exams, standardized measures of neurological impairment and functional disability, detailed pain assessment, and extensive neurophysiological testing. Through 18 months, the first two patients have been stable neurologically and the MRIs have shown evidence of solid tissue at the graft sites, without evidence of donor tissue overgrowth. Although it is still too soon to draw any firm conclusions, the findings from the initial two patients in this study suggest that intraspinal grafting of human FSC tissue is both feasible and safe.

Estrous cycle modulation of nociceptive behaviors elicited by electrical stimulation and formalin.

The impact of circulating ovarian hormones on nociceptive behaviors elicited by phasic and tonic stimuli was evaluated in rats using two behavioral tests: an operant escape task and the formalin test. The operant escape task was structured to separately evaluate hindlimb flexion reflexes, the latency of escape, and the amplitude of peak vocalization to a series of phasic electrocutaneous stimuli (0.05-0.8 mA), whereas the formalin test evaluated nociceptive behaviors elicited by tonic stimulation following a subcutaneous injection of dilute formalin (1%). Hindlimb reflex amplitude, escape latency, and peak vocalization varied across the estrous cycle, such that rats were most sensitive to electrical stimuli during proestrus (reflex and escape latency) and diestrus (vocalization). Furthermore, morphine-induced (3 mg/kg sc) attenuation of hindlimb reflex amplitude was sensitive to estrous cycling. During proestrus, morphine produced less attenuation of hindlimb reflex amplitude than during nonproestrus phases. However, estrous cycling did not alter nociceptive behaviors elicited by 1% formalin. These data support the notion that circulating ovarian hormones may differentially modulate behaviors associated with phasic and tonic pain.

Abnormal sensitization and temporal summation of second pain (wind-up) in patients with fibromyalgia syndrome.

Although individuals with fibromyalgia syndrome (FMS) consistently report wide-spread pain, clear evidence of structural abnormalities or other sources of chronic stimulation of pain afferents in the involved body areas is lacking. Without convincing evidence for peripheral tissue abnormalities in FMS patients, it seems likely that a central pathophysiological process is at least partly responsible for FMS, as is the case for many chronic pain conditions. Therefore, the present study sought to obtain psychophysical evidence for the possibility that input to central nociceptive pathways is abnormally processed in individuals with long standing FMS. In particular, temporal summation of pain (wind-up) was assessed, using series of repetitive thermal stimulation of the glabrous skin of the hands. Although wind-up was evoked both in control and FMS subjects, clear differences were observed. The perceived magnitude of the sensory response to the first stimulus within a series was greater for FMS subjects compared to controls, as was the amount of temporal summation within a series. Within series of stimuli, FMS subjects reported increases in sensory magnitude to painful levels for interstimulus intervals of 2-5 s, but pain was evoked infrequently at intervals greater than 2 s for control subjects. Following the last stimulus in a series, after-sensations were greater in magnitude, lasted longer and were more frequently painful in FMS subjects. These results have multiple implications for the general characterization of pain in FMS and for an understanding of the underlying pathophysiological basis.

The effect of maximal exercise on temporal summation of second pain (windup) in patients with fibromyalgia syndrome.

Exercise activates endogenous opioid and adrenergic systems, but attenuation of experimental pain by exercise has not been shown consistently. In this study, effects of exercise on temporal summation of late pain responses to stimulation of unmyelinated (C) nociceptors were assessed. When a preheated thermode was applied repetitively to glabrous skin of the hand in a series of brief contacts at rates of 0.2 to 0.5 Hz, the perceived intensity of late thermal sensations increased after successive contacts. This summation of pain sensations provides information regarding the status of central opioid and N-methyl-D-aspartate receptor systems. For normal subjects, temporal summation of late pain sensations was substantially attenuated when testing began 1.5 or 10 minutes after exercise. Individuals diagnosed with fibromyalgia syndrome (FMS) report generalized chronic pain that is increased after exercise. Therefore, we hypothesized that strenuous exercise would increase summation of late pain sensations in this cohort. Patients with FMS and control subjects exerted to similarly high metabolic rates, as shown by physiologic monitoring. Ratings of late pain sensations increased for patients with FMS after exercise, an effect opposite to a decrease in ratings for age/sex-matched control subjects. In contrast to this result for experimentally induced pain, clinical pain ratings were not substantially altered after strenuous exercise by patients with FMS.

Effects of systemic morphine on escape latency and a hindlimb reflex response in the rat.

The present study uses focal electrical stimulation of myelinated nociceptors to simultaneously assess behavioral responses that are organized at spinal and supraspinal sites in the rat. Hindlimb reflex amplitude and the latency to operant escape responses by a forelimb were recorded for each stimulus presentation to a hindlimb across a wide range of intensities. This paradigm provided a tool whereby effects of morphine on conscious escape responses could be delineated from effects on a segmental flexion reflex over a range of doses. Administration of morphine (3 mg/kg and 10 mg/kg, subcutaneously) increased the latency of escape responses and decreased the amplitude of reflex responses in a dose-dependent manner. However, morphine produced a greater suppression of reflex responses compared with the increase in effects on escape latencies. The effects of morphine on escape latency were not expressed at the highest stimulus intensities (0.6 to 0.8 mA), whereas reflex responses were attenuated at all suprathreshold stimulus intensities. Thus, electrically evoked, spinal-mediated responses of rats are not affected by morphine in the same manner as electrically evoked supraspinal-mediated nociceptive behaviors. However, both measures confirm evidence that responses elicited by activation of myelinated afferents are less powerfully affected by morphine than responses to input from unmyelinated nociceptors.

An operant assay of thermal pain in conscious, unrestrained rats.

Methods are described which provide quantification of learned operant and innate reflex responses to a thermal stimulus (heat or cold) and provide matched motor controls. The apparati and procedures consist of (1) an 'Escapetest' which measures latencies and durations of escape from a compartment where the floor is heated or cooled to a platform at neutral temperature in an adjacent compartment; (2) a motor and motivational control for the Escapetest, the 'Darkboxtest', which measures escape latency from bright light in a shuttle box; and (3) assessment of latencies and durations of licking or guarding responses to thermal stimulation in the absence of the escape option. Avoidance responses in the Escapetest (retreating to the escape platform in the absence of an experience of pain) are discouraged by bright illumination of the compartment containing the escape platform (brightly lit areas are aversive to rodents). Stimulus-response functions for escape from heat and cold are compared to stimulus response functions for innate lick/guard responses to the same temperatures. Substantial differences in the relationships between learned or innate responses and temperature attest to a need for methods which evaluate operant responses to nociceptive stimulation.

Effects of sacrocaudal spinal cord transection and transplantation of fetal spinal tissue on withdrawal reflexes of the tail.

Reflex responses to electrocutaneous stimulation of the tail were characterized in awake cats, before and after transection of the spinal cord at sacrocaudal levels S3-Ca1. Consistent with effects of spinal transection at higher levels, postoperative cutaneous reflexes were initially depressed, and the tail was flaccid. Recovery ensued over the course of 70-90 days after sacrocaudal transection. Preoperative and chronic postlesion reflexes elicited by electrocutaneous stimulation were graded in amplitude as a function of stimulus intensity. Chronic postlesion testing of electrocutaneous reflexes revealed greater than normal peak amplitudes, peak latencies, total amplitudes (power), and durations, particularly for higher stimulus intensities. Thus, sacrocaudal transection produced effects representative of the spastic syndrome. In contrast, exaggerated reflex responsivity did not develop for a group of cats that received transplants of fetal spinal cord tissue within sacrocaudal transection cavities at the time of injury, in conjunction with long-term immunosuppression by cyclosporine. We conclude that gray matter replacement and potential neuroprotective actions of the grafts and/or immunosuppression prevent development of the spastic syndrome. This argues that the spastic syndrome does not result entirely from interruption of long spinal pathways.