Clinical trial: multispecies probiotic supplementation alleviates the symptoms of irritable bowel syndrome and stabilizes intestinal microbiota.

BACKGROUND: Irritable bowel syndrome is the most common diagnosis in gastroenterology. Trials suggest certain probiotics to be beneficial. AIM: To investigate the effects of multispecies probiotic supplementation (Lactobacillus rhamnosus GG, L. rhamnosus Lc705, Propionibacterium freudenreichii ssp. shermanii JS and Bifidobacterium animalis ssp. lactis Bb12) on abdominal symptoms, quality of life, intestinal microbiota and inflammatory markers in irritable bowel syndrome. METHODS: Eighty-six irritable bowel syndrome patients (Rome II criteria) participated in this randomized, placebo-controlled 5-month intervention. Patients were randomized to receive daily either multispecies probiotic supplementation or placebo. Irritable bowel syndrome symptoms, quality of life, microarray-based intestinal microbiota stability (n = 20), serum cytokines and sensitive C-reactive protein were monitored. RESULTS: The composite irritable bowel syndrome score had at 5 months decreased 14 points (95% CI: -19 to -9) from baseline with the multispecies probiotic vs. three points (95% CI: -8 to 1) with placebo (P = 0.0083). Especially, distension and abdominal pain were affected. A stabilization of the microbiota was observed, as the microbiota similarity index increased with the probiotic supplementation (1.9 +/- 3.1), while it decreased with placebo (-2.9 +/- 1.7). No differences were seen in C-reactive protein. CONCLUSIONS: This multispecies probiotic seems to be an effective and safe option to alleviate symptoms of irritable bowel syndrome, and to stabilize the intestinal microbiota.

Effects of multispecies probiotic supplementation on intestinal microbiota in irritable bowel syndrome.

BACKGROUND: A multispecies probiotic has shown beneficial effects in irritable bowel syndrome. In addition, certain other probiotics have demonstrated advantageous effects, but the mechanisms behind this are poorly understood. AIM: To investigate the mode of action of a multispecies probiotic consisting of Lactobacillus rhamnosus GG, Lactobacillus rhamnosus Lc705, Propionibacterium freudenreichii ssp. shermanii JS and Bifidobacterium breve Bb99 by monitoring its effects on intestinal microbiota and markers of microbial activity. METHODS: A total of 55 irritable bowel syndrome patients participated in this placebo-controlled double-blind trial. Subjects received either multispecies probiotic or placebo supplementation daily during a 6-month period. The composition of intestinal microbiota was analysed with real-time polymerase chain reaction, short-chain fatty acids with gas chromatography and enzymes with spectrophotometer. RESULTS: Each supplemented probiotic strain was detected in faecal samples. Intestinal microbiota remained stable during the trial, except for Bifidobacterium spp., which increased in the placebo group and decreased in the probiotic group (P = 0.028). No changes in short-chain fatty acids occurred. A decrease in ss-glucuronidase activity was detected in 67% of the subjects in the probiotic group vs. 38% in the placebo group (P = 0.06). CONCLUSIONS: Factors other than the microbial groups and metabolites studied herein seem responsible for the alleviation of irritable bowel syndrome symptoms by the multispecies probiotic.

Probiotic intervention decreases serum gastrin-17 in Helicobacter pylori infection.

BACKGROUND: Previously we showed that a probiotic combination with L. rhamnosus GG was beneficial as an adjuvant therapy during H. pylori eradication. AIM: To evaluate whether probiotic combination with LGG adheres to the upper gastrointestinal mucosa and modifies H. pylori colonisation and H. pylori induced inflammation. METHODS: Thirteen patients referred for gastroduodenoscopy received a drink consisting of equal doses (2.5x10(9)CFU) of LGG, L. rhamnosus LC705, Propionibacterium freudenreichii JS and Bifidobacterium lactis Bb12 daily. Recovery of probiotics in biopsies (antrum, corpus, duodenum) and faecal samples was evaluated by strain-specific quantitative polymerase chain reaction. H. pylori colonization and gastric inflammation was investigated by urease activity ((13)C-urea breath test), histology and serum pepsinogen I, II and gastrin-17 measurements. RESULTS: Twelve patients were fully investigated; of these three of the patients had LGG adhering to the biopsies at end of the intervention. Other probiotic strains were not detected, even though the recovery of all individual probiotic strains from the faeces was significantly increased (p<0.01). After the treatment, the level of (13)C-urea breath test (p=0.063) and gastrin-17 (p=0.046) decreased. CONCLUSIONS: The decreases in (13)C-urea breath test and gastrin-17 indicate that the probiotic combination exerts a beneficial effect on gastric mucosa in H. pylori infected patients. LGG showed marginal ability to adhere to the upper gastrointestinal tract mucosa.

A probiotic mixture alleviates symptoms in irritable bowel syndrome patients: a controlled 6-month intervention.

BACKGROUND: Irritable bowel syndrome is a gastrointestinal disorder of unknown aetiology. The effect of probiotics in this syndrome remains unclear. AIM: To investigate whether a probiotic mixture containing Lactobacillus rhamnosus GG, L. rhamnosus LC705, Bifidobacterium breve Bb99 and Propionibacterium freudenreichii ssp. shermanii JS is effective in alleviating irritable bowel syndrome symptoms. METHODS: A total of 103 patients fulfilling the Rome I or II criteria took part in this 6-month, randomized, double-blind placebo-controlled trial. The patients received a probiotic capsule or a placebo capsule daily. Gastrointestinal symptoms and bowel habits were recorded. RESULTS: At the end the total symptom score (abdominal pain + distension + flatulence + borborygmi) was 7.7 (95% CI: -13.9 to -1.6) points lower in the probiotic group (P = 0.015). This represents a median reduction of 42% in the symptom score of the probiotic group compared with 6% in the placebo group. In individual symptoms, borborygmi was milder in the probiotic group (P = 0.008), and for the rest of the symptoms there was a non-significant trend. CONCLUSIONS: The results indicate that this probiotic mixture is effective in alleviating irritable bowel syndrome symptoms. Considering the high prevalence of irritable bowel syndrome and the lack of effective therapies, even a slight reduction in symptoms could have positive public health consequences.

The causal element for the lactase persistence/non-persistence polymorphism is located in a 1 Mb region of linkage disequilibrium in Europeans.

Expression of lactase in the intestine persists into adult life in some people and not others, and this is due to a cis-acting regulatory polymorphism. Previous data indicated that a mutation leading to lactase persistence had occurred on the background of a 60 kb 11-site LCT haplotype known as A (Hollox et al. 2001). Recent studies reported a 100% correlation of lactase persistence with the presence of the T allele at a CT SNP at -14 kb from LCT, in individuals of Finnish origin, suggesting that this SNP may be causal of the lactase persistence polymorphism, and also reported a very tight association with a second SNP (GA -22 kb) (Enattah et al. 2002). Here we report the existence of a one megabase stretch of linkage disequilibrium in the region of LCT and show that the -14 kb T allele and the -22 kb A allele both occur on the background of a very extended A haplotype. In a series of Finnish individuals we found a strong correlation (40/41 people) with lactose digestion and the presence of the T allele. The T allele was present in all 36 lactase persistent individuals from the UK (phenotyped by enzyme assay) studied, 31/36 of whom were of Northern European ancestry, but not in 11 non-persistent individuals who were mainly of non-UK ancestry. However, the CT heterozygotes did not show intermediate lactase enzyme activity, unlike those previously phenotyped by determining allelic transcript expression. Furthermore the one lactase persistent homozygote identified by having equally high expression of A and B haplotype transcripts, was heterozygous for CT at the -14 kb site. SNP analysis across the 1 megabase region in this person showed no evidence of recombination on either chromosome between the -14 kb SNP and LCT. The combined data shows that although the -14 kb CT SNP is an excellent candidate for the cause of the lactase persistence polymorphism, linkage disequilibrium extends far beyond the region searched so far. In addition, the CT SNP does not, on its own, explain all the variation in expression of LCT, suggesting the possibility of genetic heterogeneity.

Substance P immunoreactive sensory axons as a subset of the total axonal population in the maxillary sinus of the rabbit: a characterization of normal and infected mucosa.

Substance P (SP), one of the neuropeptides released from sensory nerves, is thought to mediate neurogenic inflammation. Although SP immunoreactive axons have been described in the sinus mucosa, no attempt has been made to characterize SP fibers as a subset of all axons present in the sinus mucosa. In addition, no study to date has characterized the changes in infected sinus mucosa. The maxillary sinus mucosa of New Zealand white rabbits was harvested from control animals and in animals with induced maxillary sinusitis. Immunohistochemical staining of the sinus mucosa for both Protein Gene Product 9.5 (PGP), a nonspecific marker for all nerves, and for SP was performed on 11 animals: 3 controls and 8 infected. In sinus mucosa from the control rabbits, <50% of all axons labeled by PGP were immunoreactive for SP. In infected mucosa, the absolute number of axons found by PGP staining decreased and nearly all of these remaining fibers were also immunoreactive for SP. We conclude that the phenotypical labeling of nerve fibers seen in normal mucosa is altered by bacterial-induced infection.

The role of pH and osmolarity in evoking the acetic acid-induced wiping response in a model of nociception in frogs.

Acetic acid applied to the hindlimb of a frog evokes a vigorous wiping of the exposed skin. The aim of this study was to determine if acetic acid evokes this wiping response by decreasing subepidermal pH. Because acetic acid is hyperosmolar, a second aim was to determine if the osmolarity of acetic acid contributed to evoking the wiping response. In behavioral experiments, different acids or acetic acid/sodium acetate buffers at different pHs were used to evoke the wiping response. In separate experiments, subepidermal pH was measured in vitro while these same solutions were applied to samples of skin from frogs. The wiping response evoked by acetic acid was associated with a decrease in subepidermal pH to a level that has been shown to activate nociceptors. Interestingly, formic, oxalic, sulfuric, and hydrochloric acid evoked the wiping response without decreasing subepidermal pH. The osmolarity of acetic acid contributed to evoking the wiping response because buffers at subthreshold pHs evoked the wiping response. Also, the osmolarity required to evoke the wiping response depended upon the pH of the buffer. Thus, acetic acid and the buffers at pH 2.97 and 4.67 could evoke the wiping response by decreasing subepidermal pH. In contrast, formic, oxalic, sulfuric, and hydrochloric acid, as well as the buffers at pH 5.17 and 5.67, evoked the wiping response through another mechanism, perhaps by increasing subepidermal osmolarity. These studies demonstrate that both pH and osmolarity may contribute to nociception produced by algesic chemicals and may be important in inflammatory pain.

SYM-2081 a kainate receptor antagonist reduces allodynia and hyperalgesia in a freeze injury model of neuropathic pain.

Cold-freeze injury at -4 degrees C to the rat sciatic nerve produces mechanical allodynia and thermal hyperalgesia [M.A. Kleive, P.S. Jungbluth, J.A. Uhlenkamp, K.C. Kajander, Cold injury to rat sciatic nerve induces thermal hyperalgesia or analgesia, 8th World Congress on Pain, Vancouver, BC, Canada, August 1996 (Abstract).]. The NMDA receptor, an excitatory amino acid (EAA) receptor, appears to be involved in the development of allodynia and hyperalgesia following nerve injury. The role, if any, of the kainate receptor, another EAA receptor, remains unknown. In the current study, we evaluated whether (2S,4R)-4-methylglutamic acid (SYM-2081), a recently developed kainate receptor antagonist, attenuates increased responsiveness following cold injury to the sciatic nerve. During baseline testing, Sprague-Dawley rats were evaluated for frequency of withdrawal from von Frey filaments and latency of withdrawal from a radiant thermal source. Animals were then anesthetized, the left sciatic nerve was exposed, and the nerve was cooled to -4 degrees C for 15 min (n=24). For control rats (n=24), all procedures were identical except that the nerve was maintained at 37 degrees C. Testing resumed on the third day following surgery. On the fifth post-operative day, SYM-2081 (150 or 100 mg/kg), fentanyl citrate (0. 04 mg/kg) or vehicle was injected intraperitoneally. Injury to the rat sciatic nerve induced a significant increase in withdrawal frequency and a significant decrease in withdrawal latency (ANOVA, p<0.05). SYM-2081 and fentanyl significantly reduced these responses (p<0.05). These results suggest that kainate and opioid receptors are involved in the mechanical allodynia and thermal hyperalgesia that develop following cold injury to the sciatic nerve.

Localized sinus inflammation in a rabbit sinusitis model induced by Bacteroides fragilis is accompanied by rigorous immune responses.

We evaluated inflammatory and immune responses against Bacteroides fragilis in a rabbit sinusitis model. Bacteroides was inoculated into the left maxillary sinus, and inflammatory (histology, cell number/cytology, lactose dehydrogenase, and apoptosis) and immune responses in the sinus, airway, and peripheral blood (PB) were determined for up to 4 weeks. In the inflamed sinus, the lactose dehydrogenase level was markedly elevated, with neutrophilic infiltration, severe tissue inflammation, and increased apoptosis. Low-grade tissue inflammation was present in the contralateral and sham-operated sinuses, but other parameters remained unchanged, and so did those in the airway and PB in the inoculated rabbits. Serum IgG antibody levels increased rapidly, were highest at 3 weeks, and began to decline at 4 weeks. Cellular immune responses (proliferation and interferon-gamma mRNA expression) against Bacteroides were detected in the PB of all inoculated rabbits. Vigorous immune responses against Bacteroides may have localized but failed to terminate inflammation in the sinus, indicating importance of microenvironmental factors.

The kainate receptor antagonist 2S,4R-4-methylglutamate attenuates mechanical allodynia and thermal hyperalgesia in a rat model of nerve injury.

Opioids and receptor antagonists of excitatory amino acids attenuate mechanical allodynia and thermal hyperalgesia in animal models of neuropathic pain. Recently, a kainate receptor antagonist, 2S,4R-4-methylglutamate, has been developed but has not been tested for antinociceptive effects in animal models of neuropathic pain. We evaluated whether 2S,4R-4-methylglutamate attenuated responses to mechanical and thermal stimuli in uninjured (control) rats and increased responsiveness in rats with chronic constriction injury. Rats were tested for a number of withdrawal responses using a calibrated von Frey filament (mechanical stimulus) and withdrawal latencies from a radiant heat source (thermal stimulus). In control rats, 2S,4R-4-methylglutamate produced a small but significant decrease in responses from the mechanical stimulus (25 mg/kg) and significantly increased withdrawal latencies from the thermal stimulus at the highest dose administered (100 mg/kg). In addition, 2S,4R-4-methylglutamate greatly attenuated increased responsiveness in rats with chronic constriction injury. At four to eight days following chronic constriction injury, animals that displayed increased responsiveness to mechanical and thermal stimuli were injected intraperitoneally with either dizocilpine maleate (0.1 mg/kg), morphine (4 mg/kg), vehicle as controls, or 2S,4R-4-methylglutamate (25, 50, 75 or 100 mg/kg). 2S,4R-4-Methylglutamate (25, 50, 75 and 100 mg/kg) significantly attenuated the frequency of responses to mechanical stimuli (Wilcoxon, P < 0.05) and the latency of responses to thermal stimuli (analysis of variance and Duncan's, P < 0.05). Dizocilpine maleate and morphine, as expected, also reduced these responses. These results suggest that, in addition to opioid and N-methyl-D-aspartate receptors, kainate receptors may play a role in the maintenance of mechanical allodynia and thermal hyperalgesia associated with peripheral nerve injury.

Middle ear pathologic changes associated with chronic anaerobic sinusitis in rabbits.

OBJECTIVE/HYPOTHESIS: To study the histopathologic changes in association with the inflammatory/immune response present in the middle ears of a rabbit model of unilateral chronic anaerobic sinusitis. STUDY DESIGN: New Zealand white rabbits, two at each experimental time point. Normal rabbits and sham-operated animals served as controls. METHODS: Left maxillary sinusitis was induced by inoculating Bacteroides fragilis surgically after closure of the ostium. Cultures, lavages, and mucosa were harvested from bilateral middle ear and sinus cavities at 1, 2, 3, and 4 weeks following inoculation. Parameters analyzed include tissue for histopathologic study, immunoglobulin G antibody (IgG Ab) against B fragilis, and lactate dehydrogenase (LDH) levels in lavage samples, interferon gamma (IFN gamma) messenger RNA (mRNA) expression in mucosal tissue, and bacterial culture. RESULTS: Despite closure of the ostium of the left sinus, mild to moderate dissemination of B fragilis into the right sinus and left and right ears were observed in some but not all rabbits (2/8, 5/7, and 2/8, respectively). Histopathologic changes in the right sinus and middle ears were much less severe in contrast to the severe inflammatory changes in the left sinus. An immune response against B fragilis appeared to occur in the sinuses and ears bilaterally independent of bacterial dissemination, as evidenced by a rise of IgG Ab in lavage fluid and detection of IFNg mRNA. Neither control nor sham-operated animals had detectable levels of IFNg mRNA or IgG Ab. In B fragilis-inoculated rabbits, the magnitude of IgG Ab responses was equivalent in the right and left ear, independent of B fragilis dissemination; IgG Ab levels in the middle ear positively correlated to each other (P < .01) and to the levels in the sinuses (P < .01 and P < .01). LDH levels were closely associated with bacterial growth and degree of tissue inflammation. CONCLUSION: This reproducible model of chronic sinusitis provides an opportunity to study the middle ear infection and inflammatory/immune responses occurring with sinusitis. Our results indicate bilateral middle ear mucosal immune responses to an elicited sinus infection, independent of B fragilis dissemination.

IgG antibody levels in the sinus, ear, and airway in a rabbit model of sinusitis with Bacteroides.

OBJECTIVE: To evaluate distribution of IgG antibodies (Ab) in the airway, ear, and sinuses in association with inflammatory changes in a rabbit sinusitis model. DESIGN: We measured IgG Ab and lactate dehydrogenase levels in solutions from sinus, airway, and middle ear lavage and in serum, and determined interferon y messenger RNA expression in sinus and ear mucosa at 1, 2, 3, and 4 weeks after inoculation with Bacteroides fragilis. SUBJECTS: Six rabbits at each time point; controls were untreated (n=5) and sham-operated rabbits at 2 and 4 weeks (n=4-5). INTERVENTION: Bacteroides fragilis was inoculated into the left maxillary sinus with ostium closed. RESULTS: IgG Ab was undetectable in all controls. IgG Ab (>50 microg/g protein) was present at 2, 3, and 4 weeks in most bilateral sinus lavage samples and in 2 of 6, 5 of 6, and 6 of 10 ear lavage samples at 2, 3, and 4 weeks, respectively, following inoculation. Inflammatory changes (histological and lactate dehydrogenase) were much greater in the inflamed sinus. IgG Ab (>50 microg/g protein) was also detected in most bronchoalveolar lavage samples after 2 weeks. Interferon gamma mRNA was undetectable in all untreated and most sham-operated controls but was detected in the bilateral sinus mucosa at 1 to 2 weeks, and remained detectable up to 4 weeks in most rabbits. Serum IgG Ab levels positively correlated with those in lavage samples, with highest correlation with right sinus lavage IgG Ab levels (r=0.56, P<.001). CONCLUSION: IgG Ab levels in the upper airway mucosa likely increase within 2 weeks following bacterial inoculation as a part of mucosal immune responses independent of tissue necrosis.

Labeling of calcitonin gene-related peptide and substance P increases in subnucleus caudalis of rabbit during maxillary sinusitis.

Calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) and substance P-like immunoreactivity (SP-LI) were evaluated in subnucleus caudalis following induction of sinusitis. Three days after induction, intensity of labeling for CGRP-LI and SP-LI increased in ipsilateral subnucleus caudalis. Labeling for CGRP-LI and SP-LI appeared normal at later time points (20 and 28 days). Early changes in these neuropeptides may contribute to the inflammatory process and painful symptoms accompanying sinusitis.

Central projections of nerves innervating the rabbit maxillary sinus localized using wheat germ agglutinin-horseradish peroxidase or choleragenoid-horseradish peroxidase.

Central projections of nerves innervating the rabbit maxillary sinus were localized by using wheat germ agglutinin-horseradish peroxidase (WGA-HRP) or choleragenoid-horseradish peroxidase (B-HRP). Tracer was placed into the left maxillary sinus; rabbits were killed 3 or 5 days later, and histochemical localization of transported WGA-HRP or B-HRP was performed. Labeled cell bodies (437-545/animal) were seen in the ipsilateral trigeminal ganglion. Very few labeled cell bodies (zero to three/animal) were observed in the contralateral ganglion. The area of cell bodies labeled by WGA-HRP appeared similar to the area of cell bodies labeled by B-HRP. Transganglionic projections from either tracer were localized to lamina II of the ipsilateral subnucleus caudalis. In addition, WGA-HRP labeling was occasionally observed in lamina I. No labeling was present in other areas of the brainstem. In contrast to the above results, other studies have demonstrated that B-HRP produces terminal-like labeling in deeper layers of the gray matter. We injected B-HRP into the infraorbital nerve and sciatic nerve, which are known to contain projections to deep layers of the gray matter. Labeling was observed in the deep layers of the medullary or spinal dorsal horn 5 days later, suggesting that nerves innervating the sinus only project to superficial laminae. These results suggest that neurons in superficial laminae of the subnucleus caudalis may be important for the reflex initiation of the increased glandular secretions in the maxillary sinus during sinusitis.

Intraplantar injection of hyaluronic acid at low pH into the rat hindpaw produces tissue acidosis and enhances withdrawal responses to mechanical stimuli.

Application of buffers covering a range of acidic pH values activates and sensitizes nociceptors and produces pain. The purpose of this study was to determine whether a range of acidic pH in tissue produces mechanical hyperalgesia. Tissue acidosis was produced in the hindpaw of the rat by intraplantar injections of hyaluronic acid (HA) adjusted to pH 7.4, 6.0, 5.0, 4.0 or 3.0. Mechanical hyperalgesia was assessed by evaluating responses to application of a von Frey monofilament to the plantar surface before and after injection of HA. In separate experiments, magnitude of tissue acidosis produced by injection of HA was determined by measuring pH of intraplantar tissue using a pH microelectrode. Although needle stick alone produced mechanical hyperalgesia, intraplantar injections of HA at pH 6.0 or 5.0 produced significantly greater mechanical hyperalgesia. In contrast, mechanical hyperalgesia produced by injection of HA at pH 7.4, 4.0 or 3.0 was not different from that produced by needle stick. Although injection of HA at low pH produced tissue acidosis in a pH dependent manner, only a narrow range of tissue acidosis (pH = 6.38-6.00) produced mechanical hyperalgesia. Our data suggest that tissue acidosis induces mechanical hyperalgesia; however, the range of tissue pH that produces this effect is limited.

Responses of cutaneous A-fiber nociceptors to noxious cold.

Responses of cutaneous nociceptors to natural stimuli, particularly mechanical and heat stimuli, have been well documented. Although nociceptors are excited by noxious cold stimuli, there have been few studies of their stimulus-response functions for cold stimuli over a wide range of stimulus temperatures. Furthermore, the proportion of nociceptors excited by noxious cold is not clear. In the present study, we examined responses of mechanosensitive A delta-nociceptors and low-threshold mechanoreceptors to a wide range of cold stimuli that included stimulus temperatures <0 degrees C. Electrophysiological recordings were made from single primary afferent fibers in the saphenous nerves of anesthetized rats. Cutaneous sensory receptors were classed according to their conduction velocity and subgrouped functionally according to their responses evoked by mechanical, heat, and cold stimuli (0 degrees C). Responses evoked by a wide range of cold stimulus intensities that included stimuli considered innocuous and noxious (painful) were then assessed. Stimuli of 20 to -20 degrees C were delivered to the receptive field via a 1-cm2 contact thermode from a base temperature of 32 degrees C. Stimuli were applied in descending order of 2 degrees C decrements. Stimulus ramp rate was 5 degrees C/s, and stimulus temperatures were applied for a duration of 10 s. A total of 90 A fibers was studied, of which 61 were nociceptors and had conduction velocity in the A delta-range (2-30 m/s). Nociceptors were classed initially as mechanical, mechanoheat, and mechanocold nociceptors. The remaining 29 fibers were low-threshold mechanoreceptors with conduction velocity in the A delta- or A beta-range (>30 m/s). These were subgrouped according to their adaptive properties as slowly or rapidly adapting, and according to whether they were excited by hair movement (hair follicle afferent fibers). All nociceptors were excited by noxious cold. Only 30% of nociceptors were considered sensitive to cold on initial classification with the use of a cold stimulus of 0 degrees C. However, all nociceptors were excited by stimulus intensities <0 degreesC. Response thresholds for cold ranged from 14 to -18 degrees C (-4.6 +/- 1.07 degrees C, mean +/- SE). The total number of impulses, discharge rate, and peak discharge increased monotonically as intensity of cold stimuli increased. Power functions were used to determine the rate at which the number of impulses increased as stimulus intensity increased. The slopes of power funcions ranged from 0.12 to 2.28 (mean 1.07 +/- 0.13). Most mechanoreceptors were not excited by cold stimuli. The only types of mechanoreceptors that responded reliably to cold stimuli were the slowly adapting mechanoreceptors. Responses usually occurred during the temperature ramp when the skin temperature was decreasing. There was no evidence that mechanoreceptors encoded the intensity of cold stimuli at intensities above or below 0 degrees C, because evoked responses did not increase with intensity of cold stimuli. It is concluded that the proportion of cutaneous A delta-nociceptors excited by noxious cold stimuli has been underestimated in previous studies. All nociceptors were excited by stimulus temperatures <0 degrees C and encoded the intensity of cold stimuli. It is therefore likely that cutaneous A delta-nociceptors contribute to the sensation of cold pain, particularly pain produced by stimulus temperatures <0 degrees C.

A nitric oxide synthesis inhibitor (L-NAME) reduces licking behavior and Fos-labeling in the spinal cord of rats during formalin-induced inflammation.

Formalin injected subcutaneously into the hindpaw of the rat produces an animal model of inflammation that exhibits a phasic component and a tonic component of pain. We evaluated the effects of a nitric oxide synthase inhibitor, N omega-nitro-L-arginine methyl ester (L-NAME), on a formalin-induced behavior, hindpaw licking, and on Fos-labeling of nuclei in the fifth lumbar spinal segment. Our results demonstrated that pretreatment with intrathecal doses of 0.3 and 1.0 mg of L-NAME significantly reduced licking behavior associated with injection of formalin into the left hindpaw of the rat. In addition, these same doses of L-NAME reduced formalin-induced Fos-labeling in the ipsilateral dorsal gray matter (as compared to the contralateral gray matter). Qualitative assessment suggested that the reduction in labeling occurred primarily in the superficial dorsal horn. The stereoisomer, D-NAME, administered at the same doses had little to no effect on either formalin-induced licking or Fos-labeling. Finally, our results revealed that total licking time was related to Fos-labeling. Rats that spent less time licking the hindpaw exhibited a smaller increase in Fos-labeling. Our results suggest that the production of nitric oxide is associated with licking behavior resulting from formalin injection into the hindpaw of rats. Our results also suggest that the production of nitric oxide and Fos are associated. Indeed, these substances may be involved in spinal pathways associated with nociception.

Excitation of rat cutaneous nociceptors by noxious cold.

Electrophysiological recordings were made from mechanosensitive nociceptors innervating hairy skin of the rat hindpaw, and responses evoked by a wide range of noxious cold stimuli (20 to -12 degrees C) were determined. All A delta and C nociceptors sampled were excited by noxious cold. Response thresholds of A delta nociceptors were significantly higher (colder stimulus temperatures) than thresholds of C fibers, and many exhibited response thresholds below 0 degree C. Responses of both classes of nociceptors increased as stimulus temperature decreased. These data demonstrate that A delta and C nociceptors are excited by a wide range of cold stimuli and suggest that the proportion of cutaneous nociceptors excited by noxious cold has been underestimated in previous studies.

Fos-like immunoreactivity increases in the lumbar spinal cord following a chronic constriction injury to the sciatic nerve of rat.

A chronic constriction injury (CCI), transection injury, or sham injury to the sciatic nerve was induced in 30 rats. Rats were then sacrificed at 1, 3, 5, 10, and 20 days following injury, and the number of cells immunohistochemically labeled for Fos-like immunoreactivity (Fos-LI) was determined in random sections from the lumbar 4 and 5 (L4 and L5) spinal segments. Non-parametric statistics (Wilcoxon) were used to compare the number of labeled cells ipsilateral to the injury to the number of labeled cells on the contralateral side. At 1 and 5 days following injury, in all treatment groups, significantly more labeled cells (P < 0.05) were observed ipsilaterally. In addition, at 3 and 10 days following injury, the CCI groups exhibited significantly more labeled cells ipsilaterally. The significant increases for the CCI groups ranged from 161% to 360%. Generally, increases were greater for the CCI groups. These results indicate that Fos-LI increases to a greater extent and for a longer duration following the CCI than following either a transection or sham injury.