• Korean Journal of Acupuncture
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• v.24 no.4
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• pp.221-231
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• 2007

목 적 : 본 연구의 목적은 시상하부에서 침처치에 대한 nitric oxide synthase (NOS)발현을 nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d)를 이용한 조직화학 염색법으로 관찰하였다. 실험방법 : 동물은 Balb/c (wild type) 와 Stat 4 knockout (KO) 생쥐를 사용하였다. 염증유도는 1% carrageenan 용액 (20ul/마리)을 발 뒤꿈치 표피에 주사하였고, 침 처치는 족삼리 (ST36)에 시침하였다. 침 처치 후 5시간까지 부종율을 부종측정기로 측정하였으며, 마지막으로 부종을 측정한 후 동물을 희생하여 뇌를 적출하여 고정하였다. 침에 대한 효과를 확인하기 위하여 NADPH-d 반응의 조직염색을 실시하였다. 염증유도와 그룹간의 유의성 검증은 one-way ANOVA를 사용하였다. 결 과 : 대조군인 Balb/c와 실험군인 stat4 KO 생쥐를 carrageenan으로 염증을 유도시에 대조군은 90%이상 유도된 반면, Stat4 KO 그룹은 50% 정도의 염증만이 유도되었다. 염증을 유도한 생쥐의 족삼리에 침 처치시 대조군은 1시간에서 약 40%정도 감소하였고 (P<0.05), Stat4 KO 실험군은 유의한 염증 감소율을 보이지 않았다. 시상하부의 lateral hypothalamic area (LHA)와 paraventricular nucleus (PVN)부위의 침에 대한 효과를 NADPH-d 에 양성으로 반응하는 세포수로 비교하여 다음과 같은 결과를 얻었다. (1) 대조군에서 염증 유도시 시상하부의 PVN는 NADPH-d 양성세포수가 감소하였고, LHA에서는 증가하였다. (2) 염증을 유도한 대조군에 침을 처치시 PVN은 세포수가 증가하였고, LHA에서는 감소하는 경향을 보였다. (3) 염증을 유도한 Stat4 KO 군에서는 시상하부의 PVN과 LHA부위 모두에서 NADPH-d 양성세포수가 감소하였고, 염증유도그룹에 침을 처치시 PVN과 LHA부위 모두에서 세포수가 증가함을 관찰 할 수 있었다. (4) 대조군과 실험군 모두에 salicylic acid로 비교하였더니 염증유도 효과 및 NADPH-d 세포 수에서 침 처치와 비슷한 결과를 나타내었다. 결 론 : 침은 염증을 유도한 생쥐에서 염증 감소에 유의한 효과가 있다. 염증을 유도한 Balb/c 와 Stat4 KO 생쥐에 침을 처치 시 시상하부의 NADPH-d 발현이 LHA부위와 PVN에서 서로 다르게 나타나는 것으로 나타난다. 이러한 현상은 침 효과가 시상하부의 위치에 대한 작용이 다르기 때문이라고 생각된다.

• Journal of Acupuncture Research
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• v.18 no.4
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• pp.116-124
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• 2001

Background and Objetive : The aim of this study was to investigate the effect of various electroacupuncture stimulation on NADPH-diaphorase in cerebral cortex, brain stem, cerebellum of spontaneously hypertensive rats. Materials and Methods : We evaluated the changes of NADPH-d-positive neurons using a histochemical method. The staining intensity of NADPH-d-positive neurons was assessed in a quantitative fashion using a microdensitometrical method based on optical density by means of an image analyzer. Results and Conculsion : The average optical density of NADPH-d-positive neurons of 100 Hz (bipolar square wave 0.2 ms duration and 100 Hz frequency) electroacupuncture treatment group significantly increased in most cortical areas comparison between the manual acupuncture and 2 Hz (bipolar square wave 0.2 ms duration and 2 Hz frequency) electroacupuncture groups. In the brain stem, the optical density of NADPH-d-positive neuron at only superficial gray layer of the superior colliculus area was same as cerebral cortex. We conclude that the morphological evidence for NADPH-d-positive neurons may be have regional change in cerebral cortex brain stem and cerebellum according to various electroacupuncture stimulations.

• Journal of Acupuncture Research
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• v.19 no.3
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• pp.95-106
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• 2002

The present study was designed to investigate the effects of various electroacupuncture(EA) stimulation on NADPH-diaphorase and Neuropeptide Y(NPY) in the cerebral cortex of Sprague-Dawley Rats. We evaluated the changes of NADPH-d-positive neurons using a histochemical method and the changes of NPY-positive neurons using a double labeling immunohistochemical method. The staining intensities of NADPH-d-positive neuron and NPY-positive neurons were assessed in a quantitative fashion using a microdensitometrical method based on optical density by means of an image analyzer. As to frequency, the optical density of NADPH-d-positive neurons of 2Hz-1 time EA group was significantly higher than that of 100Hz-1 time EA group in M1, Cg, and that of 100Hz-3 times EA group was significantly higher than that of 2Hz-3 times EA group in Vi, Au, Cg and Ins. As to treatment-time, the optical density of NADPH-d-positive neurons of 2Hz-1 time EA group was higher than 2Hz-3 times EA group in Vi, Au, Cg and Ins. And in Vi 100Hz-1 time EA group and in M1 100Hz-3 times EA group was more efficient. As to frequency, the optical density of NPY-positive neurons of 2Hz-1 time was significantly higher in Vi, and that of 100Hz-1 EA group was higher in M1. And that of 2Hz-3 times EA group was higher than 100Hz-3 times EA group in Cg. As to treatment-time, the optical density of NPY-positive neurons of 2Hz-1 time EA group was significantly higer than 2Hz-3 times EA in S1, Vi, Au. And that of 100Hz-1 time EA group was significantly higher than that of 100Hz-3 time EA in Cg. The present results demonstrated that EA changes the activity in NO and NPY system in the cerebral cortex of SHR and EA stimulation, like frequency and treatment-time, is of importance for this effect.

• Journal of Pharmacopuncture
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• v.4 no.1
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• pp.125-139
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• 2001

In order to study the effects of bee venom herbal acupuncture on the neurotransmitters of the rat brain cortex, herbal acupuncture with the bee venom group and normal saline group was performed bilaterally on the point corresponding to LI 4 of the rat. The average optical density of the neurotransmitters from the cerebral cortex was analyzed 30 minutes after the herbal acupuncture with immunohistochemical methods. The results were as follows: 1. The density of NADPH-diaphorase in the bee venom group was increased significantly at the motor cortex, visual cortex, auditory cortex, cingulate cortex, retrosplenial cortex, and perirhinal cortex, compared to the normal saline group. 2. The average optical density of vasoactive intestinal peptide in the bee venom group had significant changes at the insular cortex, retrosplenial cortex, and perirhinal cortex, compared to the normal saline group. 3. The average optical density of neuropeptide-Y in the bee venom group increased significantly at the visual cortex and cingulate cortex, compared to the normal saline group.

• The Journal of Korean Medicine
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• v.23 no.3
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• pp.26-32
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• 2002

목적 : 본 연구에서는 알코올 독성에 대하여 흰쥐의 치상회에서 새로운 신경세포의 생성 및 nitric oxide synthase (NOS) 발현에 인삼이 미치는 영향을 5-bromo-2-deoxyuridine (BrdU) 면역 조직 화학법 및 nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) 조직화학법을 통해서 관찰하고자 한다. 방법 : 실험동물을 정상군, 인삼처치군, 알코올처치군 및 알코올-인삼 처치군으로 분류하여 각각의 실험군에 3일간 BrdU (50mg/kg)를 복강주사하였다. 인삼처치군은 30mg/kg 용량의 인삼 전탕액을 중완혈에 약침주사하였고, 알코올 처치군은 2 g/kg 용량의 알코올을 투여하였으며. 알코올-인삼 처치군은 2 g/kg 용량의 알코올 및 30mg/kg 용량의 인삼 전탕액을 투여한 후 각각의 BrdU 양성 세포수와 NADPH-d 양성세포수를 관찰하였다. 결과 : 알코올 투여군은 BrdU 양성세포 및 NADPH-d 양성세포 발현이 감소하였으나, 인삼 및 알코올 인삼처치군에서는 알코올 투여군에 비해서 모두 증가하였다. 결론 : 인삼은 알코올에 의해서 유발된 새로운 신경세포 생성의 감소에 대하여 보호효과가 있으며, 알코올에 의해서 부가적으로 영향 받는 산화질소는 세포생성 조절에 중요한 역할을 하는 것으로 사려된다.

• Journal of Acupuncture Research
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• v.22 no.2
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• pp.13-18
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• 2005

This study was undertaken to investigate the effects of electroacupuncture(EA) on Choksamni(ST36), a well-known acupuncture site, on nicotinamide adenine dinucleotide phosphate-diaphorase(NADPH-d), neuropeptide Y(NPY) and vasoactive intestinal peptide(VIP) in the cerebral cortex of spontaneously hypertensive rats(SHR). EA on Choksamni was applied using 2Hz electrical biphasic pulses of 10min, 3 times a week for a total of 10 sessions. Thereafter we evaluated changes in NADPH-d-positive neurons histochemically and changes in NPY and VIP-positive neurons immunohistochemically. The optical density of NADPH-d-positive neurons in the Choksamni group was significantly lower in all areas of the cerebral cortex than in the control group. However, the optical density of NPY-positive neurons in the Choksamni group was similar to that of the controls in most areas of the cerebral cortex, with the exception of the primary motor and visual cortices. The optical density of VIP-positive neurons in the Choksamni group was significantly decreased as compared to the control group in most areas of the cerebral cortex, with the exception of the cingulate cortex. The present results demonstrated that EA on Choksamni changes the activity of the NO system, and that stimulation at the same level, causes selective changes within the peptidergic system in the cerebral cortex of SHR.

• The Korean Journal of Pain
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• v.13 no.2
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• pp.164-174
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• 2000

Background: After an injury to tissue such as the skin, hyperalgesia develops. Hyperalgesia is characterized by an increase in the magnitude of pain evoked by noxious stimuli. It has been postulated that in the mechanism of hyperalgesia (especially secondary hyperalgesia) and allodynia, a sensitization of central nervous system such as spinal dorsal horn may contribute to development of hyperalgesia. However, the precise mechanism is still unclear. In the present study, we investigated the roles of N-methyl-D-aspartate (NMDA) receptor and nitric oxide (NO) system in the mechanism of hyperalgesia, and their relations with c-fos expression Methods: Inflammation was induced by injection of complete Freund adjuvant (CFA) into unilateral hindpaw of Sprague-Dawley rat. Behavioral studies measuring paw withdrawal responses by von Frey filaments and paw withdrawal latencies by radiant heat stimuli and stainings of nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase and c-fos immunoreactivity were performed. The effects of MK-801, an NMDA receptor blocker and $N^\omega$-nitro-L-arginine (L-NNA), a nitric oxide synthase (NOS) inhibitor were evaluated. Results: 1) Injection of CFA induced mechanical allodynia, mechanical hyperalgesia and thermal hyperalgesia. And it increased the number of NADPH-diaphorase positive neurons and c-fos expression neurons. 2) MK-801 inhibited mechanical hyperalgesia and thermal hyperalgesia induced by CFA and reduced the number of NADPH-diaphorase positive neurons and c-fos expression neurons. 3) L-NNA inhibited the thermal hyperalgesia and reduced the number of NADPH-diaphorase positive neurons, but did not affect the number of c-fos expression neurons. Conclusions: These results suggest that in the mechanism of mechanical hyperalgesia, NMDA receptor but not NO-system is involved and in the case of thermal hyperalgesia both NMDA receptor and NO system are involved. NO system did not affect the expression of c-fos, but c-fos expression and NOS activity were dependent on the activity of NMDA receptor.

• 한국생물공학회:학술대회논문집
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• 2005.10a
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• pp.924-925
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• 2005

• The Korean Journal of Physiology and Pharmacology
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• v.13 no.5
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• pp.337-341
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• 2009

Signal transducer and activator of transcription 4 (STAT4), a STAT family member, mediates interleukin 12 (IL12) signal transduction. IL12 is known to be related to calorie-restricted status. In the central nervous system, IL12 also enhances the production of nitric oxide (NO), which regulates food intake. In this study, the expression of neuronal NO synthase (Nos1), which is also related to food intake, was investigated in the hypothalamic areas of Stat4 knockout (KO) mice using nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry, a marker for neurons expressing Nos1 enzyme. Western blots were also performed to evaluate Nos1 and Fos expression. Wild-type Balb/c (WT group, n=10 male) and Stat4 KO mice (Stat4 KO group, n=8 male) were used. The body weight and daily food intake in the WT group were $22.4{\pm}0.3$ and 4.4 g per day, while those in the Stat4 KO group were $18.7{\pm}0.4$ and 1.8 g per day, respectively. Stat4 mice had lower body weight and food intake than Balb/c mice. Optical intensities of NADPH-d-positive neurons in the paraventricular nucleus (PVN) and lateral hypothalamic area (LHA) of the Stat4 KO group were significantly higher than those of the WT group. Western blotting analysis revealed that the hypothalamic Nos1 and Fos expression of the Stat4 KO group was up-regulated, compared to that in the WT group. These results suggest that Stat4 may be related to the regulation of food intake and expression of Nosl in the hypothalamus.

• Journal of Acupuncture Research
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• v.21 no.2
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• pp.205-222
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• 2004

Objective: To investigate the effects of acupointed Choksamni(ST36), Kokchi(LI11) and Arbitrary acupoint on NADPH-diaphorase, neuronal nitric oxide synthase(nNOS), neuropeptide Y(NPY) and vasoactive intestinal peptide(VIP) in the cerebral cortex of spontaneously hypertensive rats. Methods: The experimental groups were divided into four groups: Normal, Choksamni(ST36), Kokchi(LI11), arbitrary group. Needles were inserted into acupoints at the depth of 0.5 cm with basic insertion method. Such stimulation was applied continuously for 10 minutes, every other day, for 10 sessions of treatments. Thereafter we evaluated changes in NADPH-d-positive neurons histochemically and changes in nNOS, NPY and VIP-positive neurons immunohistochemically. Results : The optical densities of NADPH-d-positive neurons of all the Choksamni & Kokchi groups were significantly different in all areas of cerebral cortex as compared to arbitrary group. In motor1, sensory2, cingulate2, insular, peripheral, visual cortex there was a significant difference between Choksamni & Kokchi group. The optical densities of nNOS-positive neurons of Choksamni group were significantly different in all areas except for auditory, visual and pisiform cortex and Kokchi group in all areas except for auditory and pisiform cortex as compared to arbitrary group. And there was a significant difference in cingulate1, cingulate2, ectohinal, visual cortex between Choksamni & Kokchi group. The optical densities of NPY neurons of Choksamni group were significantly different in cingulate2, insular, pisiform cortex and Kokchi group in motor1, motor2, sensory1, cingulate2, ectorhinal cortex as compared to arbitrary group. And there was no significant difference between Choksamni & Kokchi group. The optical densities of VIP neurons of Choksamni group were significantly different in all areas except for motor1, auditory cortex and Kokchi group in sensory1, insular, ectorhinal, perirhinal, visual, pisiform cortex as compared to arbitrary group. And there was a significant difference in cingulate1, cingulate2, retrosplenial, auditory corterx between Choksamni & Kokchi group. Conclusion : Our results demonstrated that acupuncture on Choksamni(ST36) & Kokchi(LI11) changes the control activities of the NO system in the cerebral cortex of SHR and according to areas there were significant difference between two groups. In all cerebral cortex areas there were distributed NPY & VIP and there were no significant difference among Choksamni(ST36), Kokchi(LI11) and arbitrary group.