Characterization of Gentamicin-Induced Apoptosis in a Cochlear Cell Model / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: Aminoglycoside antibiotics are ototoxic. Understanding of the molecular mechanisms underlying the drug-induced ototoxicity, however, has been hampered by limited cell availability. Recently, HEI-OC1 cells, which are of an immortalized cochlear cell line sensitive to ototoxic drugs, have been derived from the auditory sensory organ. This study was performed to confirm whether cultured HEI-OC1 cells can be used to evaluate aminoglycoside-induced ototoxicity and the effect of antioxidants against aminoglycoside-induced colchlear cell damage. MATERIALS AND METHOD: Gentamicin was administered for 3 days in the media containing HEI-OC1 cells. RESULTS: Cell viability was decreased by gentamicin in a dose-dependent manner. The cell number was decreased by 50% 3 days after the exposure to 2 mM gentamicin. Penicillin did not have any significant effect. Flow cytometric analysis revealed that sub G1 arrest representing cellular apoptosis was accelerated by gentamicin treatment but not by penicillin. Expression of p27Kip1, the cyclin-dependent kinase inhibitor, was exclusively increased by gentamicin. Reactive oxygen species were also increased by gentamicin when compared with those of the control or when penicillin was used. Caspase-3 activity became increased according to the elevation of gentamicin concentrations. N-acetyl cysteine, but not vitamin E or vitamin C, ameliorated cell survival dose-dependently against gentamicin. CONCLUSION:The present study reveals that the HEI-OC1 cell line is a good model to evaluate gentamicin-induced ototoxicity. The results suggest that gentamicin-induced apoptosis may be, at least partially, linked to the overproduction of a reactive oxygen species called. Nacetyl cysteine, a free radical scavenger, that decreases the gentamicin ototoxicity.

MRI Follow-up Study After Arthroscopic Repair of Multiple Rotator Cuff Tendons

PURPOSE: This study investigated the rate of retear and related factors after arthroscopic repair of rotator cuff tears involving more than one tendon. MATERIALS & METHODS: Arthroscopic repair of 22 rotator cuff tears (average size 3.2cm: average age 58 years old) involving the supraspinatus and part or all of the infraspinatus were investigated using MRI on average 10 months after repair. The status of the repaired cuff was investigated using Sugaya's classification, and the change in muscle was evaluated with Goutallier's classification. RESULTS: Retear (Sugaya grade IV, V) was found in 7cases(32%). Tears larger than 3cm had a higher retear rate(67%) than smaller tears(8%). Retear cases had Goutallier grade II or higher muscle changes preoperatively and showed aggravation of muscle atrophy postoperatively. Even without retear, reversal of muscle change was not seen CONCLUSION: Rotator cuff tears not confined to the supraspinatus had a 32% retear rate after arthroscopic repair. The size of the tear was the most crucial factor influencing retear. Retear was frequent in tear over 3cm. Atrophy of the cuff muscle worsened when the repair failed but did not improve even without retear.

Microarray Analysis of Oxygen-Glucose-Deprivation Induced Gene Expression in Cultured Astrocytes

Since astrocytes were shown to play a central role in maintaining neuronal viability both under normal conditions and during stress such as ischemia, studies of the astrocytic response to stress are essential to understand many types of brain pathology. The microarray system permitted screening of large numbers of genes in biological or pathological processes. Therefore, the gene expression patterns in the in vitro model of astrocytes following exposure to oxygen-glucose deprivation (OGD) were evaluated by using the microarray analysis. Primary astrocytic cultures were prepared from postnatal Swiss Webster mice. The cells were exposed to OGD for 4 hrs at 37 degrees C prior to cell harvesting. From the cultured cells, we isolated mRNA, synthesized cDNA, converted to biotinylated cRNA and then reacted with GeneChips. The data were normalized and analyzed using dChip and GenMAPP tools. After 4 hrs exposure to OGD, 4 genes were increased more than 2 folds and 51 genes were decreased more than 2 folds compared with the control condition. The data suggest that the OGD has general suppressive effect on the gene expression with the exception of some genes which are related with ischemic cell death directly or indirectly. These genes are mainly involved in apoptotic and protein translation pathways and gap junction component. These results suggest that microarray analysis of gene expression may be useful for screening novel molecular mediators of astrocyte response to ischemic injury and making profound understanding of the cellular mechanisms as a whole. Such a screening technique should provide insights into the molecular basis of brain disorders and help to identify potential targets for therapy.

Gene Expression Profile in Microglia following Ischemia-Reperfusion Injury

Microglial activation is thought to play a role in the pathogenesis of many brain disorders. Therefore, understanding the response of microglia to noxious stimuli may provide insights into their role in disorders such as stroke and neurodegeneration. Many genes involved in this response have been identified individually, but not systematically. In this regards, the microarray system permitted to screen a large number of genes in biological or pathological processes. Therefore, we used microarray technology to evaluate the effect of oxygen glucose deprivation (OGD) and reperfusion on gene expression in microglia under ischemia-like and activating conditions. Primary microglial cultures were prepared from postnatal mice brain. The cells were exposed to 4 hrs of OGD and 1 h of reperfusion at 37 degrees C. Isolated mRNA were run on GeneChips. After OGD and reperfusion, > 2-fold increases of 90 genes and > 2-fold decrease of 41 genes were found. Among the genes differentially increased by OGD and reperfusion in microglia were inflammatory and immune related genes such as prostaglandin E synthase, IL-1beta, and TNF-alpha. Microarray analysis of gene expression may be useful for elucidating novel molecular mediators of microglial reaction to reperfusion injury and provide insights into the molecular basis of brain disorders.

Effect of Minocycline on Nociceptive Change and Microglial Activation in a Rat Model of Sciatic Neuropathy / 대한해부학회지

Nerve injury leads to chronic neuropathic pain syndromes. Activation of microglia has been studied to investigate the role in pain development. Minocycline is known as a potent inhibitor of microglial activation in many types of the brain injury models. But it is not known whether minocycline interferes with pain and microglial activation after the peripheral nerve injury. In this study, we investigated the time course of pain and microglial activation after sciatic nerve injury and also tested the effect of minocycline using sciatic nerve ligation model. All experiments were performed using 150~180 g male Sprague-Dawley rats. The chronic constriction injury (CCI) of the sciatic nerve with four 4.0 chromic gut suture was used to induce neuropathic pain in the left sciatic nerve. The behavioral response of rats to the stimuli (heat, cold & pressure) was assessed by measuring the lifting of the foot and the avoidance of touching the floor at pre-surgical day 1, post-surgical day 1, 4, 7, and 10. The L4 ~6 spine was fixed and used to detect microglia. Oral minocycline (50 mg/kg) was administered daily to the last day of the experiment. Minocycline was administered to one group of rats from pre-surgical day 1 and minocycline treatment was initiated from post-surgical day 1, 3, and 5 in other groups. Neuropathic pain was evident from day 4 and the peak response was observed at 10 days after CCI. Minocycline significantly attenuated neuropathic pain even when treatment was delayed by 3 days. But, it had no effet when treatment initiated 5 days after injury. Minocycline also attenuated microglial activation. In summary, a correlation was evident between the neuropathic pain and microglial activation in our model and minocycline reduced both development of pain and microglial activation. Thus, minocycline can be a good candidate for the treatment of neuropathic pain. However, the administration should be initiated prior to microglial activation.

Effect of Minocycline on Activation of Glia and Nuclear Factor kappa B in an Animal Nerve Injury Model

Glial cells are activated in neuropathy and play a key role in hyperalgesia and allodynia. This study was performed to determine whether minocycline could attenuate heat hyperalgesia and mechanical allodynia, and how glial cell activation and nuclear factor kappa B (NF-kappaB) were regulated by minocycline in a model of chronic constriction of sciatic nerve (CCI). When minocycline (50 mg/kg, oral) was daily administered from 1 day before to 9 days after ligation, heat hyperalgesia and mechanical allodynia were attenuated. Furthermore, when minocycline treatment was initiated 1 or 3 days after ligation, attenuation of the hypersensitive behavior was still robust. However, the effect of attenuation was less when minocycline was started from day 5. In order to elucidate the mechanism of pain attenuation by minocycline, we examined the changes of glia and NF-kappaB, and found that attenuated hyperalgesia and allodynia by minocycline was accompanied by reduced microglial activation. Furthermore, the number of NF-kappaB immunoreactive cells increased after CCI treatment and this increase was attenuated by minocycline. We also observed translocation of NF-kappaB into the nuclei of activated glial cells. These results suggest that minocycline inhibits activation of glial cells and NF-kappaB, thereby attenuating the development of behavioral hypersensitivity to stimuli.

Effects of Central Interleukin-1 on the Cardiovascular Response in Hemorrhaged Rats

The arterial pressure is regulated by the nervous and humoral mechanisms. The neuronal regulation is mostly carried out by the autonomic nervous system through the rostral ventrolateral medulla (RVLM), a key area for the cardiovascular regulation, and the humoral regulation is mediated by a number of substances, including the angiotensin (Ang) II and vasopressin. Recent studies suggest that central interleukin-1 (IL-1) activates the sympathetic nervous system and produces hypertension. The present study was undertaken to elucidate whether IL-1 and Ang II interact in the regulation of cardiovascular responses to the stress of hemorrhage. Thus, Sprague-Dawley rats were anesthetized and both femoral arteries were cannulated for direct measurement of arterial pressure and heart rate (HR) and for inducing hemorrhage. A guide cannula was placed into the lateral ventricle for injection of IL-1 (0.1, 1, 10, 20 ng/2mul) or Ang II (600 ng/10mul). A glass microelectrode was inserted into the RVLM to record the single unit spike potential. Barosensitive neurons were identified by an increased number of single unit spikes in RVLM following intravenous injection of nitroprusside. I.c.v. IL-1beta increased mean arterial pressure (MAP) in a dose-dependent fashion, but HR in a dose-independent pattern. The baroreceptor reflex sensitivity was not affected by i.c.v. IL-1beta. Both i.c.v. IL-1alpha and beta produced similar increase in MAP and HR. When hemorrhage was induced after i.c.v. injection of IL-1beta, the magnitude of MAP fall was not different from the control. The IL-1beta group showed a smaller decrease in HR and a lower spike potential count in RVLM than the control. MAP fall in response to hemorrhage after i.c.v. injection of Ang II was not different from the control. When both IL-1 and Ang II were simultaneously injected i.c.v., however, MAP fall was significantly smaller than the control, and HR was increased rather than decreased. These data suggest that IL-1, a defense immune mediator, manifests a hypertensive action in the central nervous system and attenuates the hypotensive response to hemorrhage by interaction with Ang II.

Effects of Central GABA and Glutamate on Blood Pressure and Single Unit Spikes in the RVLM of Rats

The blood pressure (BP) is regulated by the nervous system and humoral factors, such as renin- angiotensin system, vasopressin and others. In the present study, we examined the central effects of glutamate and GABA on the cardiovascular regulation by injection of these substances into the lateral ventricle and also investigated the relationship between these central effects and the action of angiotensin II (Ang). Male Sprague Dawley rats, 350~400 g, were anesthetized with urethane and instrumented with an arterial catheter for direct measurement of BP and heart rate (HR), and an guide cannula in the lateral ventricle for drug injection. A glass microelectode was inserted into the rostral ventrolateral medulla (RVLM) for recording single unit spikes. Barosensitive neurons were identified by changes of single unit spikes in RVLM following intravenous injection of nitroprusside and phenylephrine. The effects of GABA and glutamate injected into the lateral ventricle were studied in single neuronal activity of the RVLM in addition to changes in BP and heart rate, and compared the results before and after treatment with intravenous losartan, nonpeptide Ang II-type 1 receptor antagonist (1 mg/100 g BW). Intracerebroventricular administration of GABA decreased systolic blood pressure (SBP) and HR, but increased the firing rates in the RVLM. However, intracerebroventricular glutamate injection produced effects opposite to GABA. After pretreatment of intravenous losartan, the central effects of GABA on BP and firing rate in the RVLM were significantly attenuated and that of glutamate showed a tendency of attenuation. These results suggested that central GABA and glutamate regulated BP and firing rates in RVLM were inversely related to BP change. The central effects of GABA or glutamate on the autonomic nervous function were modulated by humoral factor, Ang II, by maintaining BP.

Dual effect of dynorphin A on single-unit spike potentials in rat trigeminal nucleus

The amygdala is known as a site for inducing analgesia, but its action on the trigeminal nucleus has not been known well. Little information is available on the effect of dynorphin on NMDA receptor-mediated electrophysiological events in the trigeminal nucleus. The purpose of this study was to investigate the changes in the single neuron spikes at the trigeminal nucleus caused by the amygdala and the action of dynorphin on the trigeminal nucleus. In the present study, extracellular single unit recordings were made in the dorsal horn of the medulla (trigeminal nucleus caudalis) and the effects of microiontophoretically applied compounds were examined. When (D-Ala2, N-Me-Phe4, Glys5-ol)enkephalin (DAMGO, 10-25 mM), a mu-opioid receptor agonist, was infused into the amygdala, the number of NMDA-evoked spikes at the trigeminal nucleus decreased. However, the application of naloxone into the trigeminal nucleus while DAMGO being infused into the amygdala increased the number of spikes. Low dose (1 mM) of dynorphin in the trigeminal nucleus produced a significant decrease in NMDA-evoked spikes of the trigeminal nucleus but the NMDA-evoked responses were facilitated by a high dose (5 mM) of dynorphin. After the kappa receptors were blocked with naloxone, dynorphin induced hyperalgesia. After the NMDA receptors were blocked with AP5, dynorphin induced analgesia. In conclusion, dynorphin A exerted dose-dependent dual effects (increased & decreased spike activity) on NMDA-evoked spikes in the trigeminal nucleus. The inhibitory effect of the dynorphin at a low concentration was due to the activation of kappa receptors and the excitatory effect at a high concentration was due to activation of NMDA receptors in the trigeminal neurons.

Hemorrhage- and restraint-induced analgesia in male and female conscious rats

It is well known that stress induces analgesia. This study was designed to demonstrate the stress-induced analgesia by employing hemorrhage and restraint and to investigate its mechanism and sex difference. The degree of pain was assessed by measuring the magnitude of jaw opening reflex produced by a noxious electrical stimulation in the dental pulp and by measuring the latency to withdraw the tail from a heat ray. Restraint showed an antinociceptive response. A significant increase in pain threshold on bleeding was shown and the increase was larger in male group than in female group. The tail flick latency (TFL) on bleeding after AVP antagonist injection into the ventricle was decreased and the decrease was greater in male rats than in female rats. Castration resulted in a significant reduction of TFL. This effect was reversed by treatment with sex hormones. TFL was decreased during hemorrhage in castrated rats. This response was opposite to that in non-castrated rats. TFL was further decreased during hemorrhage after infusion of AVP antagonist, and there was a significant sex difference. These results suggest that both restraint and hemorrhage produce an antinociception and that, in hemorrhage-induced analgesia, AVP and sex hormones may play an important role and male rats show a greater analgesic response.

Effects of Intrathecal Transmitters on the Pain-induced Micturition Reflex / 대한비뇨기과학회지

PURPOSE: Irritations or painful stimuli to the bladde: may alter voiding behaviorand this may be associated with dysfunction of periurethral muscles. In our study, recording of bladder and sphincter activity in response to intravesical irritants(acetic acid, capsaicin) was done and the effects of Intrathecal administration of transmitters(enkephalin, susbtance P and its antagonist, or calcitonin gene related peptide and its antagonist) was analyzed MATERIALS AND METHODS: Adult male Sprague-Dawley rats(400-500gm) were anesthetized with intraperitoneal urethane mixed with entobar. The left femoral artery and vein were cannulated for blood pressure monitoring and drug administration. A catheter(PE 50) was inserted into the bladder dome through a midline abdominal incision and bipolar electromyographic(EMG) needle electrodes were placed into the urethral sphincter. Peak contractile pressure and EMG activities were measured on a polygraph. Saline, acetic acid, or capsaicin was infused into the bladder at 50mu1/min and saline, enkephalin (ENK), substance P(SP) and its antagonist, or calcitonin gene related peptide (CGRP) and its antagonist was administered intrathecally. RESULTS: Each micturition cycle started with an increase in bladder pressure which triggered the contraction of the external urethral sphincter. Fast Fourier transform(FFT) analysis of the external sphincter EMG revealed a peak activity at 500Hz. The inteNal and duration of EIOG bursts were 38+/-2 msec and 92+/-10 msec respectively. The interval of bladder contraction was 809+/-23 sec. Intravesical acetic acid and capsaicin activated micturition reflex earlier than in control group and intrathecal ENK, SP antagonist, and CGRP antagonist attenuated the parameters in activated micturition reflex. CONCLUSIONS: Urethral sphincte EMG showed constant burst duration and interval in spite of the infusion of acetic acid or capsaicin. Intravesical acetic acid or capsaicin increased the peak amplitude of intravesical pressure, lowered voided volume, and shortened EMG spike duration and voiding interval significantly. Intrathecal injection of ENK, SP, SP antagonist, CGRP, and CGRP antagonist changed the parameters, especially the voiding interval, voiding duration and peak pressure of the bladder.

Microinjection of glutamate into the amygdala modulates nociceptive and cardiovascular response in freely moving rats

This study was performed to examine the mean arterial pressure and nociceptive jaw opening reflex after microinjection of glutamate into the amygdala in freely moving rats, and to investigate the mechanisms of antinociceptive action of amygdala. Animals were anesthetized with pentobarbital sodium (40 mg/kg, ip). A stainless steel guide cannula (26 gauge) was implanted in the amygdala and lateral ventricle. Stimulating and recording electrodes were implanted into each of the incisor pulp and anterior digastric muscle. Electrodes were led subcutaneously to the miniature cranial connector sealed on the top of the skull with acrylic resin. After 48 hours of recovery from surgery, mean arterial pressure and digastric electromyogram (dEMG) were monitored in freely moving rats. Electrical shocks (200 musec duration, 0.5~2 mA intensity) were delivered at 0.5 Hz to the dental pulp every 2 minutes. After injection of 0.35 M glutamate into the amygdala, mean arterial pressure was increased by 8+/-2 mmHg and dEMG was suppressed to 71+/-5% of the control. Injection of 0.7 M glutamate elevated mean arterial pressure by 25+/-5 mmHg and suppressed dEMG to 20+/-7% of the control. The suppression of dEMG were maintained for 30 minutes. Naloxone, an opioid receptor antagonist, inhibited the suppression of dEMG elicited by amygdaloid injection of glutamate from 28+/-4 to 68+/-5% of the control. Methysergide, a serotonin receptor antagonist, also inhibited the suppression of dEMG from 33+/-5 to 79+/-4% of the control. However, phentolamine, an alpha-adrenergic receptor antagonist, did not affect the suppression of dEMG. These results suggest that the amygdala can modulate both cardiovascular and nociceptive responses and that the antinociception of amygdala seems to be attributed to an augmentation of descending inhibitory influences on nociceptive pathways via serotonergic and opioid pathways.

Cardiovascular Effects of Endogenous GABA in the Nucleus Tractus Solitarius

BACKGROUND: The nucleus tractus solitarius (NTS), the region of the brain stem in which primary baroreceptor afferents teminate, is critically important in the normal regulation of arterial pressure (AP). In the NTS, excitatory amino acids such as L-glutamate serve as the main neurotransmitter in the regulation of AP. However, the function of GABA in the NTS has not been established. To test the function of GABA, we applied GABAergic agents to the NTS. METHODS: The experiments were conducted on adult male Sprague-Dawley rats weighing 300-500g. A cannula (PE-50 tubing filled with heparinized saline) was inserted into the femoral artery for recording of AP and heart rate(HR). Another cannula was inserted into the femoral vein for administration of nitroprusside or phenylephrine. After rats were placed on a sterotaxic instrument, the dorsal surface of the medulla was exposed, and with the aid of a surgical microscope, the NTS was visualized. Drug injections were made into the NTS using single- or three-barreled grass micropipettes pulled to an outer diameter of 80-100(micro)m and connected to a 1(micro)l Hamilton syringe. RESULTS: The follwing results were obtained in this experiment. Injection into the NTS of 10 or 20 nmol nipecitic acid, a selective inhibitor of GABA untake, produced an increase in AP. The pressor responses evoked by two doses of nipecotic acid were not significantly different. Injection of GABA(A) agonist, musciml(5 pmol in 80 nl artificial CSF) and GABA(B) agonist, baclofen (20 pmol in 80 nl) into the NTS of urethane-anesthetized rats prodused an increase in AP of 16.6+/-1.3 and 27.6+/-1.5 mmHg, respectively. Thus the pressor response to GABA(B) agonist was greater than to GABA(A) agonist. On the other hand, microinjection of GABA(A) antagonist, bicuculline and GABA(B) antagonist, phaclofen into the NTS decreased AP by approximately 13.4+/-1.0 and 20.9+/- mmHg, respectively. Thus injection of nipecotic acid into the NTS was greater in control group compared with the muscimiol or baclofen groups. The AP changes caused by i.v. injection of nitroprusside or phenylephrine were smallest in control group and greatest in the baclofen group. When calculated as baroreflex sensitivity, the change was greatest in control group and smallest in the baclofen group. CONCLUSION: From these results it was concluded that GABA in the NTS plays an important role in the regulation of AP, especially through GABA(B) receptors, and have an inhibitory effect on baroreceptor reflex.

Oxygen Consumption at Different Treadmill Speed and Grade in Athletes and Nonathletes

This study was aimed to elucidate the relationship between combinations of treadmill speed-grade and oxygen consumption(Vo2). Twenty athetic and 20 non-athletic male college students aged 19-24yr were employed to exercise on a treadmill using 4 speeds(4.02, 5.47, 6.76 and 8.05km/h) and 5 grades(0, 8, 12, 16 and 20%). A fixed speed was selected for each session with the grade increased every 3 min. The Vo2, heart rate, stride frequency and stride length were measured during the last min of each 3-min stage. Vo2increased linearly with increasing speed and grade showing significant multiple correlations in nonathletes(Vo2=3.64x+0.831y+0.031xy-7.03, R=0.98, P<0.01) and athletes(Vo2=3.48x+0.324y+0.112xy-5.74, R=0.99, P<0.01). Stride frequency and length tended to increase with increasing speed except for the transition from walking to runnig at 8.05Km/h at which the stride frequency ran up much higher with the stride length getting lower than at 6.76Km/h. Heart rate increased linearly with increasing Vo2. The rate of increase was higher during walking than during running. These results indicate that athletes have higher rate of increase in Vo2than nonathletes at near-maximal exercise and may be used as a guideline in predicting maximum oxygen comsumption and in prescribing exercise intensity.

Effects of intracerebroventricular angiotensin II on the response to hemorrhage in conscious normotensive and hypertensive rats / 대한내분비학회지

No abstract available.

Effects of intracerebroventricular angiotensin II on the cardiovasc- ular and endocrine systems in conscius normotensive and hypertensi- ve rats / 대한내분비학회지

No abstract available.

Effect of Dibenzyline on Blood Pressure, Heart Rate snd Respiratory Rate in Rabbits During Increased Intraeranial Pressure / 대한마취과학회지

It has been reported that a rapid elevation in the intracranial pressure is associated with increased arterial pressure, reduced heart rate and irregular respiration. In the present study an effort was directed to observe the possible blocking effect of dibenzyline, an alpha-sympathetic blocking agent, administered prior to the intracranial pressure elevation. Alhino rabbits were divided into two experimental groups: in one group, the intracranial pressure was raised by infusing normal saline into the extradural space and was sustained for 30 minutes, while in the other group, dibenzyline(12mg/kg B.W.) was administered 16 hours prior to the intracanial pressure elevation. In both groups, the arterial pressure, heart rate and respiratory rate were measured for 30 minutes at 5 minuteintervals. In the intracranial pressure elevated group, the arterial pressure increased to 99 mmHg at 10 minutes in comparison with 88mmHg in the beginning. At 25 minutes, it returned to or near its initial level. The slowing of the heart rate was seen toward the end of the response, i.e. in the beginning, 269 rate/min, at the end, 214 rate/min. The respiratory rate did not show any significant change. In the dibenzyline pretreated group, the arterial pressure and heart rate did not change markedly from the initial levels. From the above result, it can be stated that dibenzyline has blocking effects on the increased arterial pressure and reduced heart rate in rabbits during increased intracranial pressure.