Subcutaneous Injection Of Capsaicin Research Articles

The role of trigeminal nucleus caudalis orexin 1 receptors in orofacial pain transmission and in orofacial pain-induced learning and memory impairment in rats

It is widely accepted that the spinal trigeminal nuclear complex, especially the subnucleus caudalis (Vc), receives input from orofacial structures. The neuropeptides orexin-A and -B are expressed in multiple neuronal systems. Orexin signaling has been implicated in pain-modulating system as well as learning and memory processes. Orexin 1 receptor (OX1R) has been reported in trigeminal nucleus caudalis. However, its roles in trigeminal pain modulation have not been elucidated so far. This study was designed to investigate the role of Vc OX1R in the modulation of orofacial pain as well as pain-induced learning and memory deficits. Orofacial pain was induced by subcutaneous injection of capsaicin in the right upper lip of the rats. OX1R agonist (orexin-A) and antagonist (SB-334867-A) were microinjected into Vc prior capsaicin administration. After recording nociceptive times, learning and memory was investigated using Morris water maze (MWM) test. The results indicated that, orexin-A (150pM/rat) significantly reduced the nociceptive times, while SB334867-A (80nM/rat) exaggerated nociceptive behavior in response to capsaicin injection. In MWM test, capsaicin-treated rats showed a significant learning and memory impairment. Moreover, SB-334867-A (80nM/rat) significantly exaggerated learning and memory impairment in capsaicin-treated rats. However, administration of orexin-A (100pM/rat) prevented learning and memory deficits. Taken together, these results indicate that Vc OX1R was at least in part involved in orofacial pain transmission and orexin-A has also a beneficial inhibitory effect on orofacial pain-induced deficits in abilities of spatial learning and memory.

Effect of pain chronification and chronic pain on an endogenous pain modulation circuit in rats

We tested the hypothesis that chronic pain development (pain chronification) and ongoing chronic pain (chronic pain) reduce the activity and induce plastic changes in an endogenous analgesia circuit, the ascending nociceptive control. An important mechanism mediating this form of endogenous analgesia, referred to as capsaicin-induced analgesia, is its dependence on nucleus accumbens μ-opioid receptor mechanisms. Therefore, we also investigated whether pain chronification and chronic pain alter the requirement for nucleus accumbens μ-opioid receptor mechanisms in capsaicin-induced analgesia. We used an animal model of pain chronification in which daily subcutaneous prostaglandin E2 (PGE2) injections into the rat’s hind paw for 14days, referred to as the induction period of persistent hyperalgesia, induce a long-lasting state of nociceptor sensitization referred to as the maintenance period of persistent hyperalgesia, that lasts for at least 30days following the cessation of the PGE2 treatment. The nociceptor hypersensitivity was measured by the shortening of the time interval for the animal to respond to a mechanical stimulation of the hind paw. We found a significant reduction in the duration of capsaicin-induced analgesia during the induction and maintenance period of persistent mechanical hyperalgesia. Intra-accumbens injection of the μ-opioid receptor selective antagonist Cys2,Tyr3,Orn5,Pen7amide (CTOP) 10min before the subcutaneous injection of capsaicin into the rat’s fore paw blocked capsaicin-induced analgesia. Taken together, these findings indicate that pain chronification and chronic pain reduce the duration of capsaicin-induced analgesia, without affecting its dependence on nucleus accumbens μ-opioid receptor mechanisms. The attenuation of endogenous analgesia during pain chronification and chronic pain suggests that endogenous pain circuits play an important role in the development and maintenance of chronic pain.

흰쥐의 구강악안면 영역에서 포르말린 통증행위반응에 TRPV1 채널의 영향

The present study investigated the inflammatory hypersensitivity following activation of the transient receptor potential vanilloid receptor-1(TRPV1) in rats. Experiments were carried out on male Sprague-Dawley rats weighing 220-260g. Following an subcutaneous injection of 5% formalin in the orofacial area, nociceptive scratching behavior was recorded for 9 successive 5-min intervals in rats. The subcutaneous injection of of 5% formalin produced noxious scratching behavior. Injection of capsaicin, TRPV1 agonist, alone into the vibrissa pad did not produced nociceptive behavior. After subcutaneous injection of capsaicin(0, ) in the formalin-treated rats, nociceptive scratching behavior was recorded for 9 successive 5-min internals. Injection of capsaicin into the vibrissa pad significantly increased the number of scratches at 1 hours after injection. Noxious behavioral responses induced by subcutaneous capsaicin injection were significantly potentiated in formalin-treated rats. Pre or post-treatment with iodo-resinaferatoxin(IRTX), TRPV1 antagonist, significantly attenuated increased nociceptive behavior. These findings suggest that activation of the TRPV1 enhanced formalin-induced inflammatory pain in the orofacial area of rats.

Sensory denervation with capsaicin reduces ovarian follicular development and delays the onset of puberty in guinea pigs

Introduction: It has been documented that mammalian ovaries receive sympathetic, parasympathetic and sensory nerve fibers. The aim of this work was to investigate the effects of sensory denervation with capsaicin at the first vaginal opening (FVO) on follicular development and the expression of TRPV1 receptors in ovary cells as well as in the dorsal root ganglia (DRGs) and lumbar dorsal spinal cord neurons of guinea pigs. The DRGs and lumbar dorsal spinal cord neurons serve as a nerve connection from the ovaries to the CNS. Materials and Methods: Female guinea pigs received a subcutaneous injection of capsaicin (30 mM) at 10 days of age (P10), while control animals were injected with vehicle. Using light microscopy, we counted healthy preantral follicles (HPF), healthy antral follicles (HAF), atretic preantral follicles (APF), and atretic antral follicles (AAF) in the ovaries at the FVO, and the numbers of TRPV1-positive cells were counted in the ovarian follicles, DRGs, and lumbar dorsal spinal cord (L2-L4) neurons by immunohistochemistry. Results: Guinea pigs treated with capsaicin showed a significant delay of FVO in comparison with the control animals (36 vs. 44 days). In the ovaries, the number of preantral and antral follicles decreased significantly. Additionally, the number of TRPV1-positive thecainterstitial cells of the antral follicles was reduced significantly, and the number of TRPV1-positive neurons in the DRGs and lumbar dorsal spinal cord (L2-L4) decreased. Thus, we showed that TRPV1 receptors throughout the sensory fibers modulate ovarian follicular development and the onset of puberty in guinea pigs. Conclusion: Sensory denervation decreases ovarian follicular development and delays the onset of puberty of guinea pigs. Our data support the idea that through TRPV1 receptors, ovarian afferent fibers sense local stimuli that are sent to the CNS.

Capsaicin-Induced Corneal Sensory Denervation and Healing Impairment Are Reversed by NGF Treatment

We aimed to evaluate the nerve growth factor (NGF) pathway and its influence on corneal healing mechanisms in normal conditions and in an animal model of corneal denervation induced by capsaicin. Peripheral sensory damage was induced in rat pups by subcutaneous injection of capsaicin and the effects evaluated by hot-plate test, corneal nerve count, and tear secretion. Corneal damage was induced in capsaicin-treated and -untreated rats by epithelial scraping. Healing rate; NGF pathway (NGF, tyrosine kinase A [TrkA], p75); and the stem cell marker p63 were evaluated by RT-PCR, ELISA, Western blot, and immunohistochemistry. The effects of exogenous NGF administration as eye drop formulation were also tested. Capsaicin treatment induced a significant reduction of peripheral sensitivity, corneal innervation, tear secretion, and corneal healing rate. The ocular effects of capsaicin treatment were associated with an NGF pathway alteration. NGF eye drop treatment aided corneal healing mechanisms through a significant increase in the NGF receptors TrkA and p75, and in the stem cell marker p63. In this study, we show that an alteration in the NGF pathway is responsible for a delay in corneal healing in an animal model of sensory denervation. Moreover, we show that NGF eye drop administration modulates corneal innervation, epithelial cell healing, and corneal stem cells. These findings may trigger further research on the role of the NGF pathway in limbal stem cell deficiency.

Electrical stimulation of deep peroneal nerve mimicking acupuncture inhibits the pressor response via capsaicin-insensitive afferents in anesthetized rats

To assess the inhibitory modulation of blood pressure by stimulation of the deep peroneal nerve (DPN) and to determine the involvement of nociceptive fibers in the modulation. All the animals were divided into six groups (A-F). The rats in groups A and B received no pretreatment. The rats in groups C and D received subcutaneous injection of capsaicin or control vehicle, respectively, near the DPN for 2 days. Those in groups E and F had the DPN exposed to capsaicin or control vehicle, respectively, for 20 min. Subsequently, pressor responses were induced by stimulation of paraventricular nucleus (PVN) either electrically (groups A and C C-F) or chemically via injection of glutamate (group B). After two stable pressor responses (baseline), all groups were subject to 5-min DPN stimulation followed by PVN stimulation for 10 s. Arterial blood pressure, heart rate, and electrocardiogram were recorded. The pressor response was calculated as the difference in the mean arterial pressure (MAP) before and after PVN stimulation, and changes from baseline in pressor response after DPN stimulation were compared between the groups. Increases of MAP of 22.88±2.18 mm Hg and 20.32±5.25 mm Hg were induced by electrical (group A) or chemical (group B) stimulation of the PVN, respectively. These pressor responses were inhibited by stimulation of the DPN, and the MAP was reduced to 12.00±2.10 mm Hg in group A (n=6, P<0.01) and 7.00±2.85 mm Hg in group B (n=6, P<0.01). Subcutaneous injection of capsaicin (125 mg/kg) near the DPN in group C (n=7) had no effect on the inhibitory effect of DPN stimulation compared with the group D (n=9), and neither did blockade of nociceptive fibers with capsaicin in group E (n=6) compared with group F (n=8). Stimulation of the DPN mimicking acupuncture has an inhibitory effect on the pressor response, and the effect is mediated by capsaicin-insensitive afferent fibers in the DPN.

Plasma corticosterone levels in mouse models of pain

Pain markedly activates the hypothalamic-pituitary-adrenal (HPA) axis and increases plasma corticosterone release interfering significantly with nociceptive behaviour as well as the mechanism of action of analgesic drugs. In the present study, we monitored the time course of circulating corticosterone in two mouse strains (C57Bl/6 and Balb/C) under different pain models. In addition, the stress response was investigated following animal handling, intrathecal (i.t.) manipulation and habituation to environmental conditions commonly used in nociceptive experimental assays. We also examined the influence of within-cage order of testing on plasma corticosterone. Subcutaneous injection of capsaicin precipitated a prompt stress response whereas carrageenan and complete Freund's adjuvant induced an increased corticosterone release around the third hour post-injection. However, carrageenan induced a longer increased corticosterone in C57Bl/6 mice. In partial sciatic nerve ligation, neuropathic pain model corticosterone increased only in the first days whereas mechanical hypersensitivity remained much longer. Animal handling also represents an important stressor whereas the i.t. injection per se does not exacerbate the handling-induced stress response. Moreover, the order of testing animals from the same cage does not interfere with plasma corticosterone levels in the intrathecal procedure. Animal habituation to the testing apparatus also does not reduce the immediate corticosterone increase as compared with non-habituated mice. Our data indicate that HPA axis activation in acute and chronic pain models is time dependent and may be dissociated from evoked hyperalgesia. Therefore, HPA-axis activation represents an important variable to be considered when designing experimental assays of persistent pain as well as for interpretation of data.

Involvement of sensory neurons in bone defect repair in rats

We investigated bone repair in sensory-denervated rats, compared with controls, to elucidate the involvement of sensory neurons. Nine-week-old male Wistar rats received subcutaneous injections of capsaicin to denervate sensory neurons. Rats treated with the same amount of vehicle served as controls. A standardized bone defect was created on the parietal bone. We measured the amount of repaired bone with quantitative radiographic analysis and the mRNA expressions of osteocalcin and cathepsin K with real-time polymerase chain reaction (PCR). Quantitative radiographic analysis showed that the standard deviations and coefficients of variation for the amount of repaired bone were much higher in the capsaicin-treated group than in the control group at any time point, which means that larger individual differences in the amount of repaired bone were found in capsaicin-treated rats than controls. Furthermore, radiographs showed radiolucency in pre-existing bone surrounding the standardized defect only in the capsaicin-treated group, and histological observation demonstrated some multinuclear cells corresponding to the radiolucent area. Real-time PCR indicated that there was no significant difference in the mRNA expression levels of osteocalcin and cathepsin K between the control group and the capsaicin-treated group. These results suggest that capsaicin-induced sensory denervation affects the bone defect repair.

Peripheral Glutamate Receptors Are Required for Hyperalgesia Induced by Capsaicin

Transient receptor potential vanilloid1 (TRPV1) and glutamate receptors (GluRs) are located in small diameter primary afferent neurons (nociceptors), and it was speculated that glutamate released in the peripheral tissue in response to activation of TRPV1 might activate nociceptors retrogradely. But, it was not clear which types of GluRs are functioning in the nociceptive sensory transmission. In the present study, we examined the c-Fos expression in spinal cord dorsal horn following injection of drugs associated with glutamate receptors with/without capsaicin into the hindpaw. The subcutaneous injection of capsaicin or glutamate remarkably evoked c-Fos expression in ipsilateral sides of spinal cord dorsal horn. This capsaicin evoked increase of c-Fos expression was significantly prevented by concomitant administration of MK801, CNQX, and CPCCOEt. On the other hand, there were not any significant changes in coinjection of capsaicin and MCCG or MSOP. These results reveal that the activation of iGluRs and group I mGluR in peripheral afferent nerves play an important role in mechanisms whereby capsaicin evokes/maintains nociceptive responses.

Autofluorescent Flavoprotein Imaging of Spinal Nociceptive Activity

Pain arises from activation of peripheral nociceptors, and strong noxious stimuli may cause an increase in spinal excitability called central sensitization, which is likely involved in many pathological pain states. So far, it has not been achieved to simultaneously visualize in vivo both the temporal and spatial aspects of spinal activity, including central sensitization. Using autofluorescent flavoprotein imaging (AFI), an optical technique suitable for mapping activity in nervous tissue, we demonstrate a close temporal and spatial correlation of electrically evoked nociceptive input with the spinal AFI signal, representing spinal neuronal activity. The AFI signal increases linearly with stimulation intensity. Furthermore, we found that the AFI signal was much larger in intensity and size when the same electrical stimulation was applied after the induction of central sensitization by a subcutaneous capsaicin injection. Finally, innocuous palpation of the hindpaw did not evoke an AFI response in naive animals, but after capsaicin injection a strong response was obtained. This is the first report demonstrating simultaneously the temporal and spatial propagation of spinal nociceptive activity in vivo.

Intrathecal substance P augments morphine-induced antinociception: Possible relevance in the production of substance P N-terminal fragments

The present study sought to examine the mechanism of substance P to modulate the antinociceptive action of intrathecal (i.t.) morphine in paw-licking/biting response evoked by subcutaneous injection of capsaicin into the plantar surface of the hindpaw in mice. The i.t. injection of morphine inhibited capsaicin-induced licking/biting response in a dose-dependent manner. Substance P (25 and 50 pmol) injected i.t. alone did not alter capsaicin-induced nociception, whereas substance P at a higher dose of 100 pmol significantly reduced the capsaicin response. Western blots showed the constitutive expression of endopeptidase-24.11 in the dorsal and ventral parts of lumbar spinal cord of mice. The N-terminal fragment of substance P (1–7), which is known as a major product of substance P by endopeptidase-24.11, was more effective than substance P on capsaicin-induced nociception. Combination treatment with substance P (50 pmol) and morphine at a subthreshold dose enhanced the antinociceptive effect of morphine. The enhanced effect of the combination of substance P with morphine was reduced significantly by co-administration of phosphoramidon, an inhibitor of endopeptidase-24.11. Administration of d-isomer of substance P (1–7), [ d-Pro 2, d-Phe 7]substance P (1–7), an inhibitor of [ 3H] substance P (1–7) binding, or antisera against substance P (1–7) reversed the enhanced antinociceptive effect by co-administration of substance P and morphine. Taken together these data suggest that morphine-induced antinociception may be enhanced through substance P (1–7) formed by the enzymatic degradation of i.t. injected substance P in the spinal cord.

S1736 EP4 Receptor Blockade Inhibits Epithelial Ion Secretion Induced By PGE2, TNFα and Il1β in Human Colon

Introduction: Increased levels of proand anti-inflammatory cytokines were observed in various segments of histologically intact small intestines in animal models of acute and chronic colitis. We recently demonstrated that these cytokines are produced de novo shortly after the induction of colitis. The trigger for this upregulation is not known. Aim: To study the possible neural involvement in the synthesis of inflammatory cytokines in remote areas from the ulcer site in rat models of colitis. Methods: Colitis was induced by rectal instillation of trinitrobenzenesulphonic acid (TNBS) or iodoacetamide in adult Sprague-Dawley rats. The capsaicin sensitive primary afferents (CSPA) were ablated using subcutaneous injections of capsaicin at time 0 (25mg/kg), 8 and 32 hours (50mg/kg). Using real time PCR, TNF-α and IL-10 mRNA expression was measured in mucosal scrapings of the duodenum, jejunum, ileum and colon at different time intervals after induction of colitis. Results: TNF-α mRNA expression increased by 3-40 times in the different intestinal segments (p<0.05) 48h after iodoacetamide induced colitis. CSPA ablation completely inhibited this upregulation in the small intestine but not in the colon. Similar results were obtained in TNBS induced colitis. Intestinal IL-10 mRNA expression increased by 6-43 times (p<0.01) 48h after iodoacetamide administration. This increase was abolished in rats subjected to CSPA ablation except in the colon where IL-10 further increased by 2 times (p<0.05). In TNBS group, there was respectively a 4-12 and 4-7 folds increase of small intestinal IL-10 mRNA expression at 1 and 21 days after colitis induction (both p<0.01). This increase was not observed in rats pretreated with capsaicin. Both capsaicin treated and untreated rats elicited a similar visual ulcer score after colitis induction. Conclusion: Intestinal CSPA fibers play a key role in the induction of a de novo synthesis of inflammatory cytokines in intestinal segments distant from the site of colitis. The study was supported by the medical practice plan (MPP) and university research board (URB), American University of Beirut-Lebanon.

S1733 The Role of the IL-6/STAT3/SOCS3 Pathway in Ulcerative Colitis Related Carcinogenesis

Introduction: Increased levels of proand anti-inflammatory cytokines were observed in various segments of histologically intact small intestines in animal models of acute and chronic colitis. We recently demonstrated that these cytokines are produced de novo shortly after the induction of colitis. The trigger for this upregulation is not known. Aim: To study the possible neural involvement in the synthesis of inflammatory cytokines in remote areas from the ulcer site in rat models of colitis. Methods: Colitis was induced by rectal instillation of trinitrobenzenesulphonic acid (TNBS) or iodoacetamide in adult Sprague-Dawley rats. The capsaicin sensitive primary afferents (CSPA) were ablated using subcutaneous injections of capsaicin at time 0 (25mg/kg), 8 and 32 hours (50mg/kg). Using real time PCR, TNF-α and IL-10 mRNA expression was measured in mucosal scrapings of the duodenum, jejunum, ileum and colon at different time intervals after induction of colitis. Results: TNF-α mRNA expression increased by 3-40 times in the different intestinal segments (p<0.05) 48h after iodoacetamide induced colitis. CSPA ablation completely inhibited this upregulation in the small intestine but not in the colon. Similar results were obtained in TNBS induced colitis. Intestinal IL-10 mRNA expression increased by 6-43 times (p<0.01) 48h after iodoacetamide administration. This increase was abolished in rats subjected to CSPA ablation except in the colon where IL-10 further increased by 2 times (p<0.05). In TNBS group, there was respectively a 4-12 and 4-7 folds increase of small intestinal IL-10 mRNA expression at 1 and 21 days after colitis induction (both p<0.01). This increase was not observed in rats pretreated with capsaicin. Both capsaicin treated and untreated rats elicited a similar visual ulcer score after colitis induction. Conclusion: Intestinal CSPA fibers play a key role in the induction of a de novo synthesis of inflammatory cytokines in intestinal segments distant from the site of colitis. The study was supported by the medical practice plan (MPP) and university research board (URB), American University of Beirut-Lebanon.

Sex differences in inflammation evoked by noxious chemical, heat and electrical stimulation

Neurogenic inflammation (NI) is a feature of several inflammatory pain conditions in which females are overrepresented. Therefore, we asked if there are sex differences in the inflammatory response evoked by well known neurogenic stimuli. We compared the amount of plasma extravasation (PE), a measure of inflammation, in the hindpaw skin of male and female rats caused by subcutaneous injection of capsaicin, application of noxious heat (51 °C water bath) or electrical stimulation of the saphenous nerve. We also compared the amount of PE in males and females evoked by substance P (SP), the principal neurogenic mediator of PE. PE was quantified using a video camera and digital image analysis to measure changes in reflectance (pixel intensity, PI) of skin due to accumulation of extravasated Evans blue (EB) dye. The increase in PI induced by capsaicin was significantly greater in females compared to males ( p < 0.001) and in estrus, diestrus, and metestrus females compared to proestrus females. The time to reach maximal capsaicin-induced PE was two times longer in estrus, diestrus, and metestrus females compared to males ( p < 0.05). PE induced by heat was also significantly greater in females compared to males ( p < 0.001), however, there was no sex-related difference in PE induced by electrical stimulation or by injection of SP. These findings show that females have a greater inflammatory response when inflammation is induced by capsaicin and noxious heat suggesting possible sex-related changes in TRPV-1 receptor mediated mechanisms. These results add to the growing list of sex difference responses to noxious somatic stimulation.

Attenuation of naloxone-induced Vc pERK hyper-expression following capsaicin stimulation of the face in aged rat

In order to clarify the effect of age-related change in trigeminal nociception, phosphorylation of extracellular signal-regulated kinase (pERK) in trigeminal spinal subnucleus caudalis (Vc) and upper cervical spinal cord neurons were studied in the aged rats following subcutaneous capsaicin injection into the whisker pad. A large number of pERK-LI cells was expressed in the superficial laminae of Vc and upper cervical spinal cord in adult and aged rats following subcutaneous capsaicin injection into the whisker pad region. The number of pERK-LI cells was largest at about 2.0mm caudal from the obex and gradually decreased in their numbers in more rostral and caudal sections. The rostro-caudal distribution profile of pERK-LI cells expressed after subcutaneous capsaicin injection into whisker pad was similar in adult and aged rats. The number of pERK-LI cells was slightly, but not significantly larger in aged rats compared with that of adults. Pretreatment with naloxone significantly increased the number of capsaicin-induced pERK-LI cells in adult rats but not in aged rats. The present findings suggest that the descending modulation system impaired with advancing age, resulting in the abnormal pain sensation in aged rats.

Final Report on the Safety Assessment of Capsicum Annuum Extract, Capsicum Annuum Fruit Extract, Capsicum Annuum Resin, Capsicum Annuum Fruit Powder, Capsicum Frutescens Fruit, Capsicum Frutescens Fruit Extract, Capsicum Frutescens Resin, and Capsaicin1

Final Report on the Safety Assessment of Capsicum Annuum Extract, Capsicum Annuum Fruit Extract, Capsicum Annuum Resin, Capsicum Annuum Fruit Powder, Capsicum Frutescens Fruit, Capsicum Frutescens Fruit Extract, Capsicum Frutescens Resin, and Capsaicin1

The Orofacial Formalin Test in the Mouse: A Behavioral Model for Studying Physiology and Modulation of Trigeminal Nociception

The aim of the current study was to adapt the orofacial formalin pain model previously developed in rats for use in mice and to characterize as fully as possible the behavioral changes in this species. The effects of subcutaneous injection of different formalin concentrations (.5%, 1%, 2%, 4%, and 8%) were examined on the face-rubbing response. In mice, formalin injection into the upper lip induced sustained face-rubbing episodes with vigorous face-wash strokes directed to the perinasal area. A positive linear relationship between formalin concentration and amplitude of the rubbing activity was observed during the first and second phase of the test with concentration up to 4%. With the highest concentration used (8%), the amplitude of both phases had plateaued. Systemic administration of morphine and paracetamol induced a dose-dependent inhibition of the rubbing behavior during the second phase. Although both paracetamol and morphine inhibited the first phase, a dose-dependent inhibition was found only for morphine. The ED50 value (95% confidence interval) for suppressing the rubbing response during the first phase was 2.45 mg/kg (1.90-3.08 mg/kg) for morphine. The ED50 values for suppressing the rubbing response during the second phase were 3.52 mg/kg (2.85-4.63 mg/kg) for morphine and 100.66 mg/kg (77.98-139.05 mg/kg) for paracetamol. Heterosegmental nociceptive stimulation evoked by subcutaneous injection of capsaicin into the back of the animal 10 min before the formalin test produced a dose-dependent inhibition of the second phase of the rubbing response. The ED50 values for suppressing the rubbing response during the first and second phases were 9.04 μg (1.36-65.13 μg) and 0.92 μg (0.28-2.99 μg), respectively. In conclusion, the mouse orofacial formalin test appears to be a reliable model for studying the behavioral encoding of the intensity of nociceptive orofacial stimulation and the counter-irritation phenomenon and for testing analgesic drugs. Perspective To further exploit the new opportunities of investigating nociceptive processing at the molecular level with the transgenic “knockout” approach, we require suitable behavioral models in mice. The presented mouse orofacial formalin test appears to be a reliable model for studying the behavioral encoding of the intensity of nociceptive stimulation and the counter-irritation phenomenon and for testing analgesic drugs.

Activation of the caspase cascade underlies the rat trigeminal primary neuronal apoptosis induced by neonatal capsaicin administration

The systemic administration of capsaicin is known to cause a massive loss of sensory primary neurons in newborn rats. Here we examined the trigeminal ganglion neurons immunohistochemically for the possible induction of activated forms of caspases-9 and -3 following a subcutaneous injection of capsaicin in newborn rats. The DNA fragmentation signal was labeled by a TUNEL method. TUNEL-positive neurons were rare (< 0.5%) at 24 h after injection of the vehicle without capsaicin. After the capsaicin injection, TUNEL-positive neurons began to increase by 12 h, reached a peak at 24 h (11.4%), and returned to the control level by 120 h. Vehicle control levels of caspase- 9-immunoreactive (ir) and caspase-3-ir neurons were low (< 0.5%). Neonatal capsaicin administration induced caspase-9-immunoreactivity (ir) and -3-ir. The temporal distributions of caspase-9-ir and caspase-3-ir neurons were similar to those of TUNEL-positive neurons with peak expressions at 24 h of 13.2 and 11.1%, respectively. A double-stain analysis at 24 h post-injection indicated 72% of TUNEL-positive neurons were caspase-9-ir, and 70% caspase-3-ir. Conversely, 78 and 68% of caspase-9-ir and caspase-3-ir neurons, respectively, were TUNEL-positive. Comparison of two adjacent sections immunostained for the two different antigens revealed the co-expression of the two caspases. These results suggest that neonatal capsaicin triggers the caspase cascade and, thereby, induces trigeminal primary neuronal apoptosis.

Postnatal development of substance P-immunoreaction in the trigeminal caudalis of neonatally capsaicin-treated mice

The trigeminal subnucleus caudalis (Vc) is a critical relay site for processing nociceptive afferent input from the orofacial area in addition to its modulation by neuroplastic change. Although an administration of capsaicin in neonates induces a selective destruction of substance P (SP)-immunoreactive nerve fibers, little information is available regarding its detailed effects on the Vc, particularly during postnatal development. The present study examined postnatal changes in the distribution of SP in the Vc and trigeminal ganglion (TG) by immunohistochemical techniques in naïve (NV) and neonatally capsaicin-treated (CP) mice, combined with a quantitative analysis. The neonatal mice received a single subcutaneous injection of capsaicin (50 mg/kg) at 48 hours after birth. The neural density of the SP-immunoreaction decreased to approximately a quarter of that in 1-week-old NV mice but increased to three-quarters of that in the NV in the superficial area after postnatal week 2. A double staining with SP and myelin basic protein confirmed the absence of any SP-immunoreaction in the myelinated nerve fibers in both NV and CP mice. The SP-immunoreaction never overlapped with non-peptidergic IB4-labeled neurons in the Vc and TG of either group. Neither the size distribution of SP-positive neurons nor their relative ratio in the TG differed between NV and CP mice at the ages of postnatal weeks 1 and 8. These findings indicate two putative origins for the emergent SP-immunoreaction in the superficial layer of the Vc of the CP mice: the surviving trigeminal neurons with SP against capsaicin treatment and/or intrinsic neurons/interneurons in the Vc without SP under normal conditions.

Sensory but not parasympathetic nerves are required for ocular vascular remodeling following chronic sympathectomy in rat

Choroidal vascularity increases following chronic sympathetic denervation in rats. The mechanisms of this remodeling are unclear. Since both nitric oxide and substance P/CGRP have been suggested as angiogenic factors in other targets, we hypothesized that sensory or parasympathetic nerves may also participate in ocular vascular remodeling. To test this hypothesis, sympathetic denervation was accomplished by superior cervical ganglionectomy. Sensory denervation was induced by subcutaneous injections of capsaicin on postnatal days 2 and 9, and ocular parasympathetic innervation was ablated by pterygopalatine ganglion excision on postnatal day 60. Eyes were processed and sectioned for light microscopic histomorphometry. Sympathetic denervation for 6 weeks resulted in increased choroidal thickness, vascular luminal area, numbers of large venules and large arterioles, and capillaries in the outer nuclear layer. Capsaicin pretreatment prevented sympathectomy-induced increases in choroidal thickness, vascular luminal area and large venules and large arterioles, whereas pterygopalatine ganglionectomy was without effect. Both sensory and parasympathetic denervation attenuated increases in outer nuclear layer capillaries. These studies indicate that increased choroidal vascularity noted after chronic sympathectomy requires intact sensory innervation.