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Objectives Patients after middle ear surgery often complain of taste disturbance and a lingual numbness. The purpose of this study was to objectively assess changes in the somatosensation of the tongue and taste function in patients undergoing stapes surgery. Study Design Prospective study. Methods Symptoms of taste disturbance and tongue numbness after surgery were investigated before and after surgery in 41 patients (13 males, 28 females; mean age 41.8 years) who underwent stapes surgery. Twenty-eight patients (9 males, 19 females; mean age 43.1 years) underwent sensory and taste function tests before and after surgery. Sensory function of the tongue was measured at the operated side and the nonoperated side using the 2-point discrimination test and an electrostimulator test. Taste function was assessed with electrogustometry (EGM). Results The chorda tympani nerve (CTN) was gently touched or stretched in all patients. Postoperative thresholds on the operated side were significantly higher than preoperative thresholds in all tests in the patients who underwent all three kinds of tests. Tongue somatosensory symptoms improved significantly earlier than the taste disturbance postoperatively, and the sensory thresholds returned to the baseline along with recovery of symptoms. Conclusion These findings suggest that dysfunction of the CTN occurred following surgery even when the CTN was preserved, and that the sensory nerve threshold of the tongue correlated with the symptom of lingual numbness. The CTN may play a role not only in taste function but also in the somatosensory function of the tongue. Level of Evidence 4. Laryngoscope, 2017

We reported differential expression of the transient receptor potential vanilloid 1 (TRPV1), the transient receptor potential ankyrin 1 (TRPA1), and the (TRPM8) in the geniculate ganglions (GGs) of naive rats. In medical practice, the chorda tympani nerve (CTN) is injured in some patients during middle-ear surgery, and results in tongue numbness and taste disorder. We investigated changes in the expression of these receptors in GGs after CTN injury. In naive-rat GGs, 11.4, 11.8, and 0.5% of neurons were found to express the TRPV1, the TRPA1, the TRPM8, respectively. At 3 days after CTN injury, 5.2 and 4.0% of activating transcription factor 3-immunoreactive neurons, considered as injured neurons, were found to express the TRPV1 and the TRPA1, respectively. Among activating transcription factor 3-immunonegative neurons, considered as uninjured neurons, 3.9 and 3.8% were found to express the TRPV1 and the TRPA1, respectively. The TRPM8 was not detected in GGs after CTN injury. We found decreased mRNA levels of the TRPV1 and the TRPA1 in all neurons, as well as in uninjured neurons of ipsilateral GGs after CTN injury. CTN injury changes the gene expression in GGs and may have effects on the tongue.

Objective Although many reports describe the short-term hearing outcomes of surgically managed labyrinthine fistulae, the long-term results remain unknown. We reviewed the long-term postoperative hearing outcomes of 14 ears of patients with cholesteatoma and labyrinthine fistulae. Methods Between 1996 and 2010, 84 patients with cholesteatoma and labyrinthine fistula underwent tympanoplasty at Hyogo College of Medicine Hospital. Fistulae were located in the lateral semicircular canal in all patients and in the superior semicircular canal in one. Fourteen patients were followed up for more than 5 years. Results The postoperative air-bone gap was ≤10 dB in one patient, between 11 and 20 dB in seven, between 21 and 30 dB in four, and ≥31 dB in two. Mean bone-conduction hearing levels on the operated side had deteriorated by 3, −1 and −2 dB at 1, 2 and 4 kHz, respectively at 1 year postoperatively, and by 8, 6 and 2 dB at 1, 2 and 4 kHz, at 5 years postoperatively. Bone-conduction hearing levels at 1 and 2 kHz were significantly deteriorated at 5 years postoperatively, compared with baseline and 1 year (P

Objective: To evaluate factors affecting the perforation closure rate for perforated otitis media. Methods: Between 2008 and 2012, a total of 118 patients with perforated chronic otitis media underwent tympanoplasty at Hyogo College of Medicine Hospital by a single surgeon. Prognostic factors were analyzed using multivariate analysis with logistic regression. Results: The success rate for tympanic perforation was 93.2%. Factors showing significance on multivariate analysis were dryness of perforation and patient age at the time of surgery. Conclusion: The results suggest that pre-surgical treatment is a key factor influencing the closure rate.

Objective To establish clinical tests for measurement of trigeminal sensitivity on the human tongue and objectively assess changes in oral trigeminal sensitivity and taste ability after chorda tympani nerve (CTN) injury. Study design Prospective study. Setting University hospital. Patients One-hundred and twenty-six patients with unilateral middle ear diseases who underwent primary middle ear surgery. Main outcomes and measures Trigeminal sensation was measured each operated side as well as nonoperated side both before and 14 days after surgery using 3 kinds of tests: Semmes-Weinstein sensory test (SW test), 2-point discrimination test, and the electrostimulator test. Taste function was assessed with electrogustometry (EGM) similarly. Results The patients which the CTN was not touched (n = 6) showed no differences between preoperative and postoperative thresholds in any tests. In the patients with sectioned CTN (n=30), postoperative thresholds on the operated side were significantly higher than preoperative thresholds on the electrostimulator test, 2-point discrimination test, and EGM. In the patients with manipulated but not sectioned CTN (n = 90), postoperative thresholds were significantly higher than preoperative thresholds on the electrostimulator test and EGM. The patients with manipulated but not sectioned CTN and abnormal EGM postoperative thresholds (n = 48) showed that postoperative thresholds were significantly higher than preoperative thresholds in all tests. Conclusion These findings suggest that the electrostimulator test was most useful to objectively assess small changes of trigeminal sensation among the 3 tests. The finding that trigeminal sensitivity of the tongue deteriorated on the operated side after CTN injury suggests that CTN function affected both taste sensation and trigeminal sensation of the tongue.

Objective The eustachian tube (ET) has two important functions, ventilation and clearance of the middle ear. We evaluated the ET ventilator function by the sonotubometry and the inflation-deflation test that are widely used today, and the ET clearance function by the saccharin test. Results of both tests were compared with surgical outcomes, and assessed which test was more closely related to the surgical outcomes. Study design Prospective case series. Setting A single university hospital. Patients and intervention One hundred four ears of 95 patients with chronic otitis media with perforation underwent type I tympanoplasty. Main outcomes and measures Based on sonotubometry and the inflation-deflation test, the patients were divided into normal, stenosis, and patulous types. Based on the saccharin test, they were divided into a normal function group and a partial/gross dysfunction group. Surgical outcomes about the hearing results and the condition of the eardrum were assessed 1 year postoperatively. Preoperative evaluation of ET function was compared with the surgical outcome. Results Sonotubometry and inflation-deflation tests showed there was no significant difference among the groups about the hearing improvement and the surgical success rate. The saccharin test showed that the rate of the hearing improvement was significantly lower in the gross dysfunction group and that the success rate of Type I tympanoplasty was significantly higher in the normal group than in the dysfunction group. Conclusion There is a relationship between the saccharin test results and surgical outcomes.

OBJECTIVES To determine the true success rate of pediatric myringoplasty. MATERIALS AND METHODS This retrospective study analyzed 67 pediatric myringoplasties performed at Hyogo College of Medicine between 2000 and 2009. We divided the pediatric population into a younger group (

Objective: To investigate the cumulative recurrence rate of pars tensa retraction cholesteatoma on the new staging system established by Japan Otological Society (JOS). Materials and Methods: It is a retrospective study and performed in a tertiary and academic center. The series comprised 71 patients with pars tensa retraction cholesteatoma who underwent surgical treatment by a single surgeon between 1994 and 2007. Results: The 10-year cumulative recurrence rate of pars tensa retraction cholesteatoma was 12.1% overall, 20.0% in Stage I, 14.4% in Stage II and 6.2% in Stage III. Conclusions: The JOS staging system for pars tensa retraction cholesteatoma did not reflect the prognosis. The development of a common international staging system for cholesteatoma in the future is desirable.

Seven hundred and eighty-four patients who underwent ossiculoplasty performed by a single surgeon were reviewed in this paper. We previously reported a similar study in another journal using the 1995 American Academy of Otolaryngology Head and Neck Surgery guidelines. In the present paper, hearing outcomes were estimated based on the 2000 Japan Otological Society guidelines. The prognostic factors were analyzed using a multivariate analysis with logistic regression. Hearing outcomes were successful in 517 patients (65.9%). Type III tympanoplasty, stapes with normal mobility, children, patients without cholesteatoma and ossiculoplasty with non-cartilaginous materials were significantly favorable predictive factors. A multivariate analysis should be performed to investigate prognostic factors of ossiculoplasty. Better knowledge of these predictive factors contributes to the surgeon's judgment and the information given to patients.

Most of the clinical cases experienced taste disturbance after stapes surgery, and in a few cases this disturbance persisted for a long time. The animal experiment suggested the role of geniculate ganglion (GG) cells in nerve generation.To clinically examine taste disorder and its recovery after stapes surgery and experimentally demonstrate a role of GG.Taste function after preservation of chorda tympani nerve (CTN) in stapes surgery was prospectively investigated with a questionnaire and electrogustometry (EGM). Further, expression of neurotrophic factors in GG after injury of CTN was examined by in situ hybridization histochemistry (ISSH) and RT-PCR.Among the cases, 15/18 (83.3%) were associated with taste disturbance and 6/18 (33.3%) were associated with tongue numbness 2 weeks after surgery; however, the symptoms ceased in 14/18 cases (77.8%). Two weeks after surgery, the EGM threshold was found to be elevated in 15/18 cases (83.3%), while in 10/18 cases (55.6%), it did not decrease until 1 year after surgery. Expression of ISSH and amplified bands of BDNF and GFR increased at 7 and 14 days after nerve injury in ipsilateral GGs and also increased at 7 days on the contralateral side.

Toll-like receptors (TLRs) play an essential role in innate immune responses and in the initiation of adaptive immune responses. Microglia, the resident innate immune cells in the CNS, express TLRs. In this study, we show that TLR3 is crucial for spinal cord glial activation and tactile allodynia after peripheral nerve injury. Intrathecal administration of TLR3 antisense oligodeoxynucleotide suppressed nerve injury-induced tactile allodynia, and decreased the phosphorylation of p38 mitogen-activated protein kinase, but not extracellular signal-regulated protein kinases 1/2, in spinal glial cells. Antisense knockdown of TLR3 also attenuated the activation of spinal microglia, but not astrocytes, caused by nerve injury. Furthermore, down-regulation of TLR3 inhibited nerve injury-induced up-regulation of spinal pro-inflammatory cytokines, such as interleukin-1beta, interleukin-6, and tumor necrosis factor-alpha. Conversely, intrathecal injection of the TLR3 agonist polyinosine-polycytidylic acid induced behavioral, morphological, and biochemical changes similar to those observed after nerve injury. Indeed, TLR3-deficient mice did not develop tactile allodynia after nerve injury or polyinosine-polycytidylic acid injection. Our results indicate that TLR3 has a substantial role in the activation of spinal glial cells and the development of tactile allodynia after nerve injury. Thus, blocking TLR3 in the spinal glial cells might provide a fruitful strategy for treating neuropathic pain.

Mitogen-activated protein kinase (MAPK) plays an important role in the induction and maintenance of neuropathic pain. Transforming growth factor-activated kinase 1 (TAK1), a member of the MAPK kinase kinase family, is indispensable for the activation of c-Jun N-terminal kinase (JNK) and p38 MAPK. We now show that TAK1 induced in spinal cord astrocytes is crucial for mechanical hypersensitivity after peripheral nerve injury. Nerve injury induced a striking increase in the expression of TAK1 in the ipsilateral dorsal horn, and TAK1 was increased in hyperactive astrocytes, but not in neurons or microglia. Intrathecal administration of TAK1 antisense oligodeoxynucleotide (AS-ODN) prevented and reversed nerve injury-induced mechanical, but not heat hypersensitivity. Furthermore, TAK1 AS-ODN suppressed the activation of JNK1, but not p38 MAPK, in spinal astrocytes. In contrast, there was no change in TAK1 expression in primary sensory neurons, and TAK1 AS-ODN did not attenuate the induction of transient receptor potential ion channel TRPV1 in sensory neurons. Taken together, these results demonstrate that TAK1 upregulation in spinal astrocytes has a substantial role in the development and maintenance of mechanical hypersensitivity through the JNK1 pathway. Thus, preventing the TAK1/JNK1 signaling cascade in astrocytes might provide a fruitful strategy for treating intractable neuropathic pain. © 2008 Wiley-Liss, Inc.

Heat and cold hyperalgesia is a common feature of inflammatory pain. To investigate whether activation of extracellular signal-regulated protein kinase 5 (ERK5), also known as big mitogen-activated protein kinase 1, in primary sensory neurons participates in inflammatory pain, we examined the phosphorylation of ERK5 in the dorsal root ganglion (DRG) after peripheral inflammation. Inflammation induced by complete Freund's adjuvant produced heat and cold hyperalgesia on the ipsilateral hind paw and induced an increase in the phosphorylation of ERK5, mainly in tyrosine kinase A-expressing small- and medium-size neurons. In contrast, there was no change in ERK5 phosphorylation in the spinal dorsal horn. ERK5 antisense, but not mismatch, oligodeoxynucleotide decreased the activation of ERK5 and suppressed inflammation-induced heat and cold hyperalgesia. Furthermore, the inhibition of ERK5 blocked the induction of transient receptor potential channel TRPV1 and TRPA1 expression in DRG neurons after peripheral inflammation. Our results show that ERK5 activated in DRG neurons contribute to the development of inflammatory pain. Thus, blocking ERK5 signaling in sensory neurons that has the potential for preventing pain after inflammation.

Neuropathic pain that occurs after peripheral nerve injury is poorly controlled by current therapies. Increasing evidence shows that mitogen-activated protein kinase (MAPK) play an important role in the induction and maintenance of neuropathic pain. Here we show that activation of extracellular signal-regulated protein kinases 5 (ERK5), also known as big MAPK1, participates in pain hypersensitivity caused by nerve injury. Nerve injury increased ERK5 phosphorylation in spinal microglia and in both damaged and undamaged dorsal root ganglion (DRG) neurons. Antisense knockdown of ERK5 suppressed nerve injury-induced neuropathic pain and decreased microglial activation. Furthermore, inhibition of ERK5 blocked the induction of transient receptor potential channels and brain-derived neurotrophic factor expression in DRG neurons. Our results show that ERK5 activated in spinal microglia and DRG neurons contributes to the development of neuropathic pain. Thus, blocking ERK5 signaling in the spinal cord and primary afferents has potential for preventing pain after nerve damage.

Alterations in the intracellular signal transduction pathway in primary afferents may contribute to pain hypersensitivity. Recently, we have reported that the phosphorylation of extracellular signal-regulated protein kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) occurs in primary afferent neurons in response to noxious stimulation of the peripheral tissue, i.e., activity-dependent activation of ERK1/2 and p38 MAPK in dorsal root ganglion (DRG) neurons. In the present study, we investigated the phosphorylation of ERK5, also known as big MAPK1, in the DRG by noxious stimulation using immunohistochemistry. Capsaicin injection induced phosphorylated ERK5 (p-ERK5) in small-to-medium diameter sensory neurons with a peak at 2 min after capsaicin injection. Furthermore, we examined the p-ERK5 labeling in the DRG after noxious heat and cold stimuli and found a stimulus intensity-dependent increase in the number of activated neurons. Most of these p-ERK5-immunoreactive neurons were small- and medium-sized neurons, which coexpressed transient receptor potential (TRP) ion channel TRPV1 and TRPA1 after noxious heat and cold stimuli, respectively. In contrast, there was no change in ERK5 phosphorylation in the spinal dorsal horn. The i.t. administration of ERK5 antisense oligodeoxynucleotide reversed heat hyperalgesia, but not mechanical allodynia, produced by capsaicin injection. Taken together, these findings suggest that the in vivo activation of the ERK5 signaling pathway in sensory neurons by noxious stimulation may be, at least in part, correlated with functional activity and, further, involved in the development of pain hypersensitivity.

The p75 neurotrophin receptor (p75NTR) has been implicated in diverse neuronal responses, including survival, cell death, myelination, and inhibition of regeneration. However, the role of p75NTR in neuropathic pain, for which there is currently no effective therapy, has not been explored. Here, we report that the pharmacological blockade of p75NTR in primary sensory neurons reversed neuropathic pain after nerve injury. Nerve injury increased the expression and axonal transport of p75NTR and phosphorylation of TrkA in the uninjured primary afferents. Functional inhibition of p75NTR suppressed injury-induced neuropathic pain and decreased the phosphorylation of TrkA and p38 mitogen-activated protein kinase, and the induction of transient receptor potential channels in dorsal root ganglion (DRG) neurons. Our results show that p75NTR induced in undamaged DRG neurons facilitates TrkA signaling and contributes to heat and cold hyperalgesia.

Patients with neuropathic pain frequently experience hypersensitivity to cold stimulation. However, the underlying mechanisms of this enhanced sensitivity to cold are not well understood. After partial nerve injury, the transient receptor potential ion channel TRPV1 increases in the intact small dorsal root ganglion (DRG) neurons in several neuropathic pain models. In the present study, we precisely examined the incidence of cold hyperalgesia and the changes of TRPA1 and TRPM8 expression in the L4 and L5 DRG following L5 spinal nerve ligation (SNL), because it is likely that the activation of two distinct populations of TRPA1- and TRPM8-expressing small neurons underlie the sensation of cold. We first confirmed that L5 SNL rats developed cold hyperalgesia for more than 14 days after surgery. In the nearby uninjured L4 DRG, TRPA1 mRNA expression increased in trkA-expressing small-to-medium diameter neurons from the 1st to 14th day after the L5 SNL. This upregulation corresponded well with the development and maintenance of nerve injury-induced cold hyperalgesia of the hind paw. In contrast, there was no change in the expression of the TRPM8 mRNA/protein in the L4 DRG throughout the 2-week time course of the experiment. In the injured L5 DRG, on the other hand, both TRPA1 and TRPM8 expression decreased over 2 weeks after ligation. Furthermore, intrathecal administration of TRPA1, but not TRPM8, antisense oligodeoxynucleotide suppressed the L5 SNL-induced cold hyperalgesia. Our data suggest that increased TRPA1 in uninjured primary afferent neurons may contribute to the exaggerated response to cold observed in the neuropathic pain model.

The roles of capsaicin, menthol, and mustard oils and their receptors in geniculate ganglion (GG) neurons still remain to be elucidated. These receptors belong to the transient receptor potential (TRP) family. Capsaicin-, menthol-, and mustard oil-sensitive receptors are TRPV1, TRPM8, and TRPA1, respectively. The present study aimed to investigate the expression of TRPV1, TRPM8, and TRPA1 in naive rat GG neurons. Furthermore, we examined whether these TRP-expressing GG neurons are myelinated A-fiber or unmyelinated C-fiber neurons. Firstly, using reverse transcription-polymerase chain reaction, TRPV1 mRNA and TRPA1 mRNA were distinctly detected in the naive GG. TRPM8 mRNA was faintly detected. Secondly, using in situ hybridization, TRPV1 mRNA- or TRPA1 mRNA-labeled neurons (signal/noise ratioor= 10) were observed in 15-20% of GG neurons. Few neurons were labeled by TRPM8 mRNA. Thirdly, neurofilament 200 (NF200) protein, a marker of mylinated A-fiber neurons, was detected in 57% of naive GG neurons. Coexpression of TRPV1 mRNA or TRPA1 mRNA with NF200 was detected in 10% of GG neurons. The present study confirmed the expression of the TRP receptors in the naive GG. The possible roles of TRP receptors in naive GG neurons in somatosensory or gustatory function were suggested.

OBJECTIVE To evaluate the results of bilateral same-day surgery for bilateral perforated chronic otitis media in comparison with our previous report. STUDY DESIGN Prospective study. PATIENTS Seventeen patients ranging in age from 5 to 70 years (mean, 52.5 yr), with bilateral perforated chronic otitis media. METHODS Two hundred cases of chronic otitis media were operated on at the Department of Otolaryngology, Hyogo College of Medicine from December 1998 to November 2002. Fifty-nine patients (29.5%) had bilateral disease and 17 patients (8.5%) underwent bilateral same-day surgery. RESULTS The postoperative air-bone gap was less than 10 dB on both sides in 5 of 17 (29%) and less than 20 dB in 15 of 17 (88%). The postoperative hearing level was less than 20 dB in both ears in 4 of 17 (24%), less than 30 dB in 7 of 17 (41%), and less than 40 dB in 10 of 17 (59%). The rate of closure of the ear drum was 20 of 22 (91%) in the simple underlay myringoplasty series and 9 of 12 (75%) in the conventional myringoplasty/tympanoplasty series. The rate of closure of the ear drum on both sides was successful in 4 of 5 (80%) that underwent simple underlay myringoplasty/simple underlay myringoplasty and 8 of 12 (67%) that underwent simple underlay myringoplasty/conventional myringoplasty/tympanoplasty, whereas that on at least one side was successful in 100%. CONCLUSION Bilateral same-day surgery for bilateral perforated chronic otitis media is possible if operative indications are considered. Furthermore, it will help patients save time and money.

Cold hyperalgesia is a well-documented symptom of inflammatory and neuropathic pain; however, the underlying mechanisms of this enhanced sensitivity to cold are poorly understood. A subset of transient receptor potential (TRP) channels mediates thermosensation and is expressed in sensory tissues, such as nociceptors and skin. Here we report that the pharmacological blockade of TRPA1 in primary sensory neurons reversed cold hyperalgesia caused by inflammation and nerve injury. Inflammation and nerve injury increased TRPA1, but not TRPM8, expression in tyrosine kinase A-expressing dorsal root ganglion (DRG) neurons. Intrathecal administration of anti-nerve growth factor (anti-NGF), p38 MAPK inhibitor, or TRPA1 antisense oligodeoxynucleotide decreased the induction of TRPA1 and suppressed inflammation- and nerve injury-induced cold hyperalgesia. Conversely, intrathecal injection of NGF, but not glial cell line-derived neurotrophic factor, increased TRPA1 in DRG neurons through the p38 MAPK pathway. Together, these results demonstrate that an NGF-induced TRPA1 increase in sensory neurons via p38 activation is necessary for cold hyperalgesia. Thus, blocking TRPA1 in sensory neurons might provide a fruitful strategy for treating cold hyperalgesia caused by inflammation and nerve damage.

Objective: Although many studies report short-term hearing results after surgically managing semicircular canal fistulae, long-term results are still unknown. In the present study, long-term postoperative hearing outcomes were reviewed in 14 ears of patients with cholesteatoma and semicircular canal fistulae.Method: Between 1996 and 2010, 84 patients with cholesteatoma and semicircular canal fistulae underwent tympanoplasty at Hyogo College of Medicine Hospital. The location of the fistula was the lateral semicircular canal in all cases and superior semicircular canal in 5 cases. Fourteen patients were followed up for more than 5 years.Results: Postoperative air-bone gap was ≤10 dB in 1 case, between 11 and 20 dB in 7 cases, between 21 and 30 dB in 4 cases, and ≥31 dB in 2 cases. Mean bone-conduction hearing levels on the operated side had deteriorated by 3 dB in 1 kHz, by −1 dB in 2 kHz, and −2 dB in 4 kHz at 1 year postoperatively and by 8 dB, 6 dB, and 2 dB at 5 years postoperatively, respectively. Bon...

OBJECTIVE To identify differences in taste function among patients with chronic otitis media, cholesteatoma, and noninflammatory disease before middle ear surgery. STUDY DESIGN Retrospective analysis. SETTING Tertiary referral hospital. PATIENTS Two hundred thirty ears underwent primary middle ear surgery from January 2006 to December 2010. The subjects consisted of 84 men and 146 women with ages ranging from 20 to 60 years (mean, 43.9 yr). There were 95 ears with chronic otitis media, 61 ears with pars flaccida retraction-type cholesteatoma, 22 ears with pars tensa retraction-type cholesteatoma, and 52 ears with noninflammatory diseases such as otosclerosis and ossicular anomalies. MAIN OUTCOME MEASURE The patients underwent taste testing using electrogustometry and the filter paper disk method in the regions controlled by the chorda tympani nerve. RESULTS None of the patients complained of taste dysfunction before middle ear surgery. Among the 4 groups, the patients with pars tensa retraction-type cholesteatoma displayed the highest electrogustometry threshold. The filter paper disk method did not detect any significant differences among the 4 groups. CONCLUSION Our findings suggest that the filter paper disk scores of all taste qualities (sweet, salty, sour, and bitter tastes) are not deteriorated preoperatively during chronic inflammation in the middle ear and that taste function is affected most in patients with pars tensa retraction-type cholesteatoma.

Heat and cold hyperalgesia is a common feature of inflammatory pain. To investigate whether activation of extracellular signal-regulated protein kinase 5 (ERK5), also known as big mitogen-activated protein kinase 1, in primary sensory neurons participates in inflammatory pain, we examined the phosphorylation of ERK5 in the dorsal root ganglion (DRG) after peripheral inflammation. Inflammation induced by complete Freund's adjuvant produced heat and cold hyperalgesia on the ipsilateral hind paw and induced an increase in the phosphorylation of ERK5, mainly in tyrosine kinase A-expressing small- and medium-size neurons. In contrast, there was no change in ERK5 phosphorylation in the spinal dorsal horn. ERK5 antisense, but not mismatch, oligodeoxynucleotide decreased the activation of ERK5 and suppressed inflammation-induced heat and cold hyperalgesia. Furthermore, the inhibition of ERK5 blocked the induction of transient receptor potential channel TRPV1 and TRPA1 expression in DRG neurons after peripheral inflammation. Our results show that ERK5 activated in DRG neurons contribute to the development of inflammatory pain. Thus, blocking ERK5 signaling in sensory neurons that has the potential for preventing pain after inflammation.

Hypersensitivity to mechanical stimulation is a well documented symptom of neuropathic pain, for which there is currently no effective therapy. Src-family kinases (SFKs) are involved in proliferation and differentiation and in neuronal plasticity, including long-term potentiation, learning, and memory. Here we show that activation of SFKs induced in spinal cord microglia is crucial for mechanical hypersensitivity after peripheral nerve injury. Nerve injury induced a striking increase in SFK phosphorylation in the ipsilateral dorsal horn, and SFKs were activated in hyperactive microglia but not in neurons or astrocytes. Intrathecal administration of the Src-family tyrosine kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) suppressed nerve injury-induced mechanical hypersensitivity but not heat and cold hypersensitivity. Furthermore, PP2 reversed the activation of extracellular signal-regulated protein kinase (ERK), but not p38 mitogen-activated protein kinase, in spinal microglia. In contrast, there was no change in SFK phosphorylation in primary sensory neurons, and PP2 did not decrease the induction of transient receptor potential ion channel TRPV1 and TRPA1 in sensory neurons. Together, these results demonstrate that SFK activation in spinal microglia contributes to the development of mechanical hypersensitivity through the ERK pathway. Therefore, preventing the activation of the Src/ERK signaling cascade in microglia might provide a fruitful strategy for treating neuropathic pain.

Two cold-sensitive transient receptor potential (TRP) channels, TRPA1 and TRPM8, have been identified and considered interesting because of their possible roles in thermosensation, nociception and other functions. Recently, we have reported that the phosphorylation of extracellular signal-regulated protein kinase and p38 mitogen-activated protein kinase occurred in primary afferent neurons in response to noxious heat stimulation of the peripheral tissue, i.e. activity-dependent activation of extracellular signal-regulated protein kinase and p38 in dorsal root ganglion neurons. In the present study, we investigated the phosphorylation of extracellular signal-regulated protein kinase, p38, and c-Jun N-terminal kinase in the rat dorsal root ganglion by cold stimulation using immunohistochemistry. Cold stimuli (28-4 degrees C) were applied by immersion of the hind paw into a water bath (six times of 10 s stimulation and 10 s interval, total 2 min). Noxious cold stimulation induced phosphorylated-extracellular signal-regulated protein kinase and phosphorylated-p38, but not phosphorylated-c-Jun N-terminal kinase, in small to medium diameter sensory neurons with a peak at 2 min after stimulation. We found that a cold stimulation at 4 degrees C showed a marked increase in the number of activated neurons. Furthermore, double staining for phosphorylated-extracellular signal-regulated protein kinase and phosphorylated-p38 showed no colocalization in the dorsal root ganglion neurons. We then performed double-labeling experiments for TRPA1 and TRPM8 mRNA and phosphorylation of mitogen-activated protein kinase. The majority of phosphorylated-extracellular signal-regulated protein kinase-positive neurons also expressed TRPM8 mRNA, whereas phosphorylated-p38 heavily colocalized with TRPA1 mRNA after noxious cold stimulation. Our data suggest that the noxious, but not innocuous, cold stimulation in vivo induced differential activation of extracellular signal-regulated protein kinase and p38 pathways in each subpopulation containing TRPA1 or TRPM8 in dorsal root ganglion.

A number of rat neuropathy models have been developed to simulate human neuropathic pain conditions, such as spontaneous pain, hyperalgesia, and allodynia. In the present study, to determine the relative importance of injury site (proximal or distal to the primary afferent neurons) and injury type (motor or sensory), we examined pain-related behaviors and changes of brain-derived neurotrophic factor expression in the dorsal root ganglion in sham-operated rats, and in the L5 dorsal rhizotomy, L5 ventral rhizotomy, L5 dorsal rhizotomy+ventral rhizotomy, and L5 spinal nerve transection models. L5 ventral rhizotomy and spinal nerve transection produced not only mechanical and heat hypersensitivity, but also an increase in brain-derived neurotrophic factor mRNA/protein in the L5 dorsal root ganglion at 7 days after surgery. In contrast, rats in the L5 dorsal rhizotomy and dorsal rhizotomy+ventral rhizotomy groups did not show both pain behaviors at 7 days after surgery, despite brain-derived neurotrophic factor upregulation in medium- and large-size neurons in the L5 dorsal root ganglion. On the other hand, L5 spinal nerve transection, but not dorsal rhizotomy, dorsal rhizotomy+ventral rhizotomy or ventral rhizotomy, increased the expression of brain-derived neurotrophic factor in the L4 dorsal root ganglion at 7 days after surgery. Taken together, these findings suggest that the upregulation of brain-derived neurotrophic factor expression in the L4 and L5 dorsal root ganglion neurons may be, at least in part, involved in the pathophysiological mechanisms of neuropathic pain and that the selective nerve root injury models may be useful for studying the underlying mechanisms of chronic pain after nerve injury.

To investigate whether activation of mitogen-activated protein kinase (MAPK) in damaged and/or undamaged primary afferents participates in neuropathic pain after partial nerve injury, we examined the phosphorylation of extracellular signal-regulated protein kinase (ERK), p38 MAPK, and c-Jun N-terminal kinase (JNK) in the L4 and L5 dorsal root ganglion (DRG) in the L5 spinal nerve ligation (SNL) model. We first confirmed, using activating transcription factor 3 and neuropeptide Y immunoreactivity, that virtually all L4 DRG neurons are spared from axotomy in this model. In the injured L5 DRG, the L5 SNL induced the activation of ERK, p38, and JNK in different populations of DRG neurons. In contrast, in the uninjured L4 DRG, the L5 SNL induced only p38 activation in tyrosine kinase A-expressing small- to medium-diameter neurons. Intrathecal ERK, p38, and JNK inhibitor infusions reversed SNL-induced mechanical allodynia, whereas only p38 inhibitor application attenuated SNL-induced thermal hyperalgesia. Furthermore, the L5 dorsal rhizotomy did not prevent SNL-induced thermal hyperalgesia. We therefore hypothesized that p38 activation in the uninjured L4 DRG might be involved in the development of heat hypersensitivity in the L5 SNL model. In fact, the treatment of the p38 inhibitor and also anti-nerve growth factor reduced SNL-induced upregulation of brain-derived neurotrophic factor and transient receptor potential vanilloid type 1 expression in the L4 DRG. Together, our results demonstrate that the L5 SNL induces differential activation of MAPK in injured and uninjured DRG neurons and, furthermore, that MAPK activation in the primary afferents may participate in generating pain hypersensitivity after partial nerve injury.