延髓内脏带至杏仁核投射通路参与迷走神经刺激抑制癫痫发作的研究

目的 观察迷走神经→延髓内脏带(MVZ)→杏仁中央核的儿茶酚胺能通路是否参与了迷走神经刺激(vagus nerve stimulation,VNS)抑制癫痫的调节；是否存在由迷走神经→延髓内脏带→海马的直接投射参与抑痫。方法 将逆行追踪剂WGA—HRP注入大鼠—侧杏仁中央核或腹侧海马，48h后，给予迷走神经刺激，观察MVZ内WGA—HRP逆行标记的细胞、Fos蛋白、TH阳性神经元的表达及分布。结果 杏仁核注射组大鼠MVZ内可见HPR／Fos／TH二重标记的细胞；海马注射组MVZ内未见HRP逆标神经元，但HRP逆行标记与Fos阳性双重标记细胞出现存隔区和下丘脑室旁该。结论 提示迷走神经→延髓内脏带→杏仁中央核的投射通路直接参与VNS抑痫过程，而且与儿茶酚胺能神经元有关；迷走神经→延髓内脏带→隔区、下丘脑室旁核中继至海马的间接通路也参与了抑痫。     

Noxious somatic stimulation-induced expression of Fos-like immunoreactivity in catecholaminergic neurons with habenular nucleus projection in the medullary visceral zone of rat

In order to study the expression of Fos protein in catecholaminergic neurons in the medullary visceral zone (MVZ), which project to the habenular nucleus (HB), a triple-labeling method combining wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) retrograde tracing with anti-Fos and anti-tyrosine hydroxylase (TH) immunohistochemical staining was used in the rat. WGA-HRP was stereotaxically injected into unilateral HB. Forty-eight hours later, 50 μl of 8% formalin was injected into the foot pad of the right front paw. Two hours after formalin injection, animals were anesthetized and perfused transaortically. Coronal sections (40 μm) were cut from the cervical segment of spinal cord, the medulla oblongata and WGA-HRP injected area with a cryostat. First, sections of the injected area and the medulla oblongata were histochemically processed to demonstrate the presence of retrogradely transported WGA-HRP using the chromogen tetramethylbenzidine (TMB). Then sections of the spinal cord and the medulla oblongata were immunostained with anti-Fos and anti-TH antibodies using the ABC method. Under the light microscope, seven types of variously labeled neurons could be identified in MVZ, namely Fos and TH-immunoreactive (Fos- or TH-IL) neurons, WGA-HRP labeled ones, Fos/HRP, Fos/TH and HRP/TH double-labeled and Fos/HRP/TH triple-labeled cells. The results suggest that some catecholaminergic neurons in MVZ could send projections to HB and this pathway may be involved to relay nociceptive information from spinal cord to brainstem and on to the forebrain.     

Visceral noxious stimulation induced expression of Fos protein in medullary catecholaminergic neurons projecting to nucleus accumbens in the rat: a study with triple labeling method of HRP tracing combined with Fos and TH immunohistochemistry

Horseradish peroxidase (HRP) was stereotaxically injected into the nucleus accumbens (Acb), and visceral noxious stimulation given by injecting formalin into the stomach. Sections of the medulla were subjected to HRP reaction combined with immunohistochemical reactions for Fos protein (ABC method) and tyrosine hydroxylase (TH, PAP method). The catecholaminergic neurons of the medulla (including vagal complex, ventrolateral medulla and reticular formation between them) which expressed Fos protein and projected to Acb were studied. The results showed that HRP retrogradely labeled cells were seen in the medulla bilaterally with apparent ipsilateral predominance and TH-LI and Fos-LI single labeled cells were bilaterally distributed; HRP/TH,TH/Fos double labeled neurons were more numerous than HRP/Fos double-labeled neurons. HRP/TH/Fos triple-labeled neurons were small in number and were mainly distributed in the nucleus tractus solitarii (nTS) and ventrolateral medulla (VLM), but only a few labeled cells were located in RF between nTS and VLM. It is concluded that TH-LI neurons in the medulla projected to Acb and some of them expressed Fos protein after noxious stimulation of the stomach.     

大鼠延髓内脏带与下丘脑室旁核、视上核往返投射通路参与高渗调节反应的研究

目的探讨延髓内脏带(MVZ)与下丘脑室旁核(PVN)和视上核(SON)之间是否存在往返渗透压投射通路。方法通过给予大鼠饮用3%氯化钠的方法制作高渗刺激模型,并用WGA-HRP逆行追踪、抗Fos、抗酪氨酸羟化酶(TH)或加压素(VP)及胶质纤维酸性蛋白(GFAP)免疫组织化学相结合的四重标记方法,观察MVZ、PVN和SON中WGA-HRP、Fos、TH、VP和GFAP阳性分布及表达状况。结果高渗刺激后MVZ、PVN和SON内Fos阳性细胞明显增多;GFAP阳性结构也明显增多,其分布与Fos阳性细胞分布基本一致,表现为胞体肥大、突起粗长。星形胶质细胞(AST)紧密包绕在神经元周围形成神经元-AST复合体(N-ASC)。结论神经元和AST以N-ASC的形式共同参与渗透压调节反应,体内存在MVZ和SON或PVN之间往返的渗透压调节通路。     

Flurothyl-induced seizures in rats activate Fos in brainstem catecholaminergic neurons

Autonomic changes accompany seizures in both animals and humans. While ictal autonomic dysfunction can be life-threatening, the participating neural networks involved are poorly understood. In this study we examined the activation of Fos following generalized seizures in brainstem structures known to mediate autonomic function. Adult female rats were sacrificed 2 h after flurothyl-induced seizures. Double-immunostaining for c-Fos and dopamine-beta-hydroxylase (DBH), and c-Fos and phenylethanol-N-methyl-transferase (PNMT) were performed in brainstem slices. Numbers of DBH-labeled neurons expressing Fos-like immunoreactivity (FLI) (DBH/Fos) and PNMT labeled neurons expressing FLI (PNMT/Fos) were counted in the noradrenergic (A1, A2, A5, A7) and adrenergic (C1, C2) cell groups localized in pons and medulla oblongata. Among the experimental animals, the highest degree of co-localization of DBH/Fos neurons was observed in the locus coeruleus (A6; 87.7%), and in the A1(72.8%) cell group located in the caudal ventrolateral medulla (VLM). No co-localization of DBH/Fos neurons was observed in control animals. The highest degree of co-localization of PNMT/Fos neurons was observed in the C1 adrenergic cell group (84.2%) located in the rostral VLM. Control animals showed very few (5.5%) PNMT/Fos co-localized neurons in the C1 adrenergic cell group. Our results indicate that flurothyl-induced generalized seizures in rats activate catecholaminergic neurons in the pons and medulla oblongata. Further studies are necessary to determine whether activation of brainstem catecholaminergic neurons contribute to the autonomic manifestations that frequently accompany epileptic seizures.     

Mating-activated brainstem catecholaminergic neurons in the female rat

Central catecholaminergic systems play an important role in the control of reproductive activities including sexual behavior, luteinizing hormone (LH) and prolactin secretion. It has been reported that catecholaminergic neurons in the locus coeruleus (A6) are activated by mating in rabbits and ferrets, animals known as reflex ovulators. This study used Fos as a marker of neuronal activity to examine whether brainstem catecholaminergic neurons are activated by mating in the spontaneous ovulator, the female rat. Proestrous rats receiving intromissions (mated group) from males or mounts-without-intromission (mounted group) were sacrificed along with rats taken directly from their home cage (control group) 90 min after the beginning of mating or mounting. Double-label immunocytochemistry was used to examine the expression of c-Fos in catecholaminergic neurons labeled by tyrosine hydroxylase (TH) antibody, or adrenergic neurons labeled by phenylethanolamine-N-methyl transferase (PNMT) antibody. Double label immunofluorescent immunohistochemistry was used to determine the number of neurons containing the estrogen receptor (ERalpha) that were activated by mating in these brain areas. The results showed that mating-with-intromissions induced a significant increase in the percentage of TH/Fos colabeled neurons in both A1 and A2 cells compared to mounting-without-intromission or control. In both these areas, over 50% ERalpha-ir neurons were activated after mating while mounting-without-intromission did not affect the percentage of colabeled Fos/ERalpha neurons. In A6 region, neither the expression of Fos nor the percentage of TH/Fos colabeled cells was influenced by either mating or mounting compared to controls. The percentage of PNMT-containing neurons colabeled with Fos was not different in C1 and C2 among the three experimental groups. The results indicate that catecholaminergic neurons were activated by mating in A1 and A2 but not in adjoining adrenergic C1 and C2 cells. In contrast to the findings that catecholaminergic neurons in A6 are activated by mating in induced ovulators, mating did not affect neuronal activity in A6 neurons in the female rat. In A1 and A2 areas, a high percentage of neurons containing ERalpha were activated by mating suggesting both tactile and hormonal information may converge on these populations of neurons. The activated catecholaminergic neurons in A1 and A2 may be an important pathway by which sensory information generated during sexual interaction modulates both behavior and pituitary function.     

大鼠蛛网膜下腔出血致多器官功能障碍综合征延髓内脏带FOS蛋白的表达及迷走神经的作用研究

目的 观察大鼠蛛网膜下腔出血(SAH)致多器官功能障碍综合征(MODS)并/不并迷走神经切断时延髓内脏带(MVZ)FOS蛋白的表达规律,探讨MVZ和迷走神经在SAH致MODS中的可能调控机制.方法 Willis环注血法建立SAH致MODS模型,膈下迷走神经切断法阻断迷走神经,免疫组织化学法检测MVZ内FOS蛋白的表达.结果 (1)SAH组和SAH+SDV组MVZ内FOS阳性表达明显高于正常对照组、假手术组、SDV组(P<0.01).但SAH+SDV组明显少于SAH组(P<0.01),两组FOS蛋白表达均在24h达峰.(2)SAH组和SAH+SDV组各时相点各脏器存在不同程度的炎性损害,以24～36h病理改变最显著,与FOS蛋白的表达峰值一致,而且SAH+SDV组的炎性损害较SAH组更明显、持久.结论 (1)SAH后各周围脏器的炎性改变与MVZ内FOS蛋白的表达规律相一致,提示MVZ参与了SAH后周围脏器功能的调控,是SAH致MODS的直接调控中枢之一;(2)膈下迷走神经切断增加了SAH后MODS的发生率,增强了SAH所致的周围脏器的炎性损害,提示迷走神经在SAH致MODS时对周围脏器具有潜在的保护作用.

Abstract：

Objective To investigate FOS protein expression in the medullary visceral zone( MVZ) of the rats following subarachnoid hemorrhage ( SAH) complicated with multiple organ dysfunction syndrome ( MODS ) which complicated or uncomplicated by vagotomy,and to discuss the possible pathogenesis of MVZ and vagus nerve in the rats following SAH complicated with MODS. Methods SAH was induced in rats by injecting arterial blood through the circle of willis. The vagus never was blocked in rats from subdlaphragamatic vagotomy. The area density of positive staining expressing FOS protein were analyzed for the relative content of immunohistochemistry. Results (1) In the MVZ, the expression in FOS protein in SAH and SAH + SDV groups were all obviously more than control group, sham-operative group and SDV group ( P < 0. 01). But the expression in SAH + SDV group were fewer and sparser than SAH group( P < 0. 01) . FOS protein expressed in SAH and SAH + SDV groups showed time progress and reached peak at 24h after SAH. (2) After SAH there are various inflammatory lesions on the organ tissue at every time point in SAH group and SAH + SDV group. The organ has significantly pathological changes at 24 ～ 36h point which is at equal pace the regularity of the expression of FOS protein. But the inflammatory lesions in SAH + SDV group was more serious and persistent than that in SAH group. Conclusions ( 1) There was the consistency between FOS protein expression and histological changes of each organ after SAH. It cued that MVZ may be one of the direct regulative centre after SAH by MODS. ( 2 ) Subdlaphragamatic vagotomy can add the incidences of MODS after SAH and can aggravate the imflamming lesions of peri-organs. Vagus never can protect peri-organs potatially when SAH complicated by MODS.     

Specificity and generality of the involvement of catecholaminergic afferents in hypothalamic responses to immune insults

Catecholamine-containing projections from the medulla have been implicated in the mediation of activational responses of the paraventricular nucleus of the hypothalamus (PVH) provoked by moderate doses of interleukin-1 (IL-1). To test the generality of this mechanism, rats bearing unilateral transections of aminergic projections were challenged with intravenous IL-1 (2 microg/kg), bacterial lipopolysaccharide (LPS; 0.1, 2.0, or 100 microg/kg), or saline and perfused 3 hours later; their brains were then prepared for quantitative analysis of Fos induction and relative levels of corticotropin-releasing factor (CRF) mRNA. LPS provoked a robust and dose-related increase in Fos expression within the PVH on the intact side of the brain at all doses tested; the response to IL-1 approximated that to the lowest LPS dose. On the lesioned side, Fos induction was significantly reduced at all dosage levels but was eliminated only at the lowest dosage. The percentage reduction was greatest (75%) in IL-1-challenged rats and was progressively less in animals treated with increasing LPS doses (67, 59, and 46%, respectively). Specificity of aminergic involvement was tested by using intra-PVH administration of the axonally transported catecholamine immunotoxin, antiDBH-saporin. This treatment abolished IL-1-induced elevations of Fos-ir and CRF mRNA in the PVH but left intact comparable responses to restraint stress. These data support a specific involvement of ascending catecholaminergic projections in mediating PVH responses to IL-1 and LPS. Residual Fos induction seen in lesioned animals in response to higher doses of LPS provides a basis for probing additional circuits that may be recruited in a hierarchical manner in response to more strenuous or complex immune insults.     

Effect of indomethacin on the c-fos expression in AVP and TH neurons in rat brain induced by lipopolysaccharide

The aim of the present study was to investigate the effect of indomethacin on the Fos expression in arginine vasopressin (AVP)-containing neurons in the hypothalamus and tyrosine hydroxylase (TH)-containing neurons in the locus coeruleus (LC) using dual-labeled immunohistochemistry. In the hypothalamus, intraperitoneal (i.p) injection of different doses [2.5 microg/100 g, 125 microg/100 g body weight (b.w.)] of lipopolysaccharide (LPS) induced a significant Fos expression in AVP neurons in the supraoptic nucleus (SON), the magnocellular division (mPVN) and the parvocellular division (pPVN) of the paraventricular nucleus (PVN). Pretreatment with the cyclooxygenase inhibitor indomethacin (0.8 mg/100 g b.w.) significantly blocked the Fos expression in these AVP neurons induced by a low dose of LPS (2.5 microg/100 g) but had no effect on the Fos expression induced by a high dose of LPS (125 microg/100 g). Similarly, in the brain stem, a large number of TH-positive neurons in the LC expressed Fos after administration of either dose of LPS. Indomethacin prevented the Fos expression induced only by a low dose of LPS, but not by a high dose of LPS. These results suggest that the activation of AVP neurons in PVN and SON and TH neurons in LC response to immune challenge might be mediated-at least partially-by prostaglandins.     

Fetal hypothalamic transplants promote survival and functional regeneration of axotomized adult supraoptic magnocellular neurons

This study investigated the mechanisms by which fetal hypothalamic transplants promote functional recovery in neurohypophysectomized rats. Seven days after neurohypophysectomy (resulting in urine osmolalities of about 800 mOsm), young adult male Long-Evans rats received either fetal hypothalamic grafts (n = 10) or sham transplants (n = 7). Recovery from the lesioned-induced diabetes insipidus was monitored for 6 months and then the transplant sites were evaluated by immunocytochemistry. Surviving host supraoptic magnocellular neurons and neurophysin-positive grafted neurons were counted and their formation of neurohemal contacts evaluated by retrograde transport of systemically injected horseradish peroxidase (HRP). There were significantly more surviving supraoptic magnocellular neurons in neurohypophysectomized animals with median eminence-placed grafts (2236 +/- 261 neurons/animal) than in animals with ectopic tissue grafts (895 +/- 142 neurons/animal) or sham implants (1052 +/- 92 neurons/animal). Almost all surviving host magnocellular neurons were labeled with retrogradely transported HRP while virtually none of the grafted neurophysin positive cells showed evidence of HRP uptake. The degree of functional recovery was directly correlated with the increased survival of host neurons. By 8 weeks post-transplantation, animals with median eminence-placed grafts had recovered from their diabetes insipidus and could concentrate their urine to within normal limits (2,120 +/- 110 mOsm). This recovery was stable for the remainder of the 6 month test period. In contrast, animals with ectopic grafts and sham transplants had permanent deficits in fluid regulation. Our results provide evidence for the long-term capacity of fetal neural tissue implants to rescue host neurons from the cell death that typically occurs in the mature central nervous system after axotomy.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acute audiogenic stress-induced activation of CRH neurons in the hypothalamic paraventricular nucleus and catecholaminergic neurons in the medulla oblongata

Strong c-fos expression was induced in neuronal cells of several brain nuclei and the auditory cortex by a short duration auditory stimulation (white noise) in rats. By double immunostaining, Fos-immunoreactive cell nuclei appeared in corticotropin-releasing hormone (CRH)-containing neurons in the hypothalamic paraventricular nucleus, but not in CRH neurons elsewhere in the brain including the central nucleus of the amygdala. Among brain catecholaminergic neurons, only cells in the medulla oblongata (in the A1/C1and A2/C2 cell groups) established double immunostaining for Fos and tyrosine hydroxylase. Sound stimulus in rats with unilateral tympanotomy and plugging the airways resulted in side differences of Fos immunoreactivity in neurons of the auditory pathways and the auditory cortex, but the effect was bilateral in hypothalamic and amygdaloid nuclei. The present data provide evidence for the participation of CRH-synthesizing neurons in hypothalamus and medullary catecholaminergic neurons in the central organization of responses to audiogenic stress stimuli.     

Differential effects of reserpine on brainstem catecholaminergic neurons revealed by Fos protein immunohistochemistry.

The effects of a single systemic injection of reserpine on c-fos proto-oncogene expression in catecholaminergic neurons of the rat brainstem were studied by immunohistochemistry for Fos proteins (Fos). In control rats, a few Fos immunoreactive neuronal nuclei were observed in the tectum and mesencephalic central gray. Within hours after drug injection, a substantial number of brainstem neurons stained intensely for Fos. The staining was maximal at 6 h and returned to control levels within 24 h. Double-immunohistochemical staining with antibodies to tyrosine hydroxylase revealed that in all noradrenergic (NA) neuron subgroups except the A2 group, the majority of NA neurons stained for Fos. Most adrenergic neurons were also labeled. In contrast, aside from some cells in the ventral tegmental area, reserpine did not induce Fos immunoreactivity in dopaminergic neurons. Numerous non-catecholaminergic neurons were intensely stained with Fos in the substantia nigra pars reticulata, ventral tegmental area, mesencephalic central gray, pontine nuclei and tectum. A small number of Fos immunoreactive neurons was also observed in raphe nuclei. Injection of saline (i.p.) resulted in a moderate increase in Fos immunoreactivity in the locus ceruleus, in A1/C1 neurons and in the mesencephalic central gray. The results demonstrate that acute reserpine treatment induces Fos expression in distinct populations of brainstem neurons, comprising both catecholaminergic and non-catecholaminergic neurons. Thus, induction of Fos by reserpine does not coincide with the site of action of this drug. The distribution of Fos immunoreactive NA neurons after reserpine treatment is comparable to that reported after application of stressful stimuli.(ABSTRACT TRUNCATED AT 250 WORDS)     

Involvement of medullary A2 noradrenergic neurons in the activation of oxytocin neurons after conditioned fear stimuli

Fear-related stimuli activate oxytocin neurons in the hypothalamus and facilitate oxytocin release from the pituitary. Oxytocin neurons in the supraoptic nucleus receive direct noradrenergic innervations from the A1 and A2 cell groups in the medulla oblongata. In the present study, we investigated the role of hypothalamic-projecting noradrenergic neurons in controlling oxytocin cell activity following fear-related stimuli in rats. An unconditioned fear stimulus (intermittently applied footshock) or conditioned fear stimulus induced expression of Fos protein, a protein product of an immediate-early gene, in magnocellular oxytocin neurons in the supraoptic or paraventricular nucleus. A neurotoxin, 5-amino-2,4-dihydroxy-alpha-methylphenylethylamine, microinjected into the vicinity of the supraoptic nucleus, selectively depleted the noradrenaline contents of the nucleus and blocked the Fos expression in the supraoptic nucleus after the unconditioned or conditioned fear stimulus. In the medulla oblongata, the unconditioned fear stimulus induced expression of Fos protein in both A2/C2 and A1/C1 catecholaminergic neurons. On the other hand, the conditioned fear stimulus induced expression of Fos protein preferentially in the A2/C2 neurons. Furthermore, the unconditioned fear stimulus induced Fos expression in the A1/C1 and A2/C2 catecholaminergic neurons labelled with retrograde tracers previously injected into the supraoptic nucleus. The conditioned fear stimulus induced Fos expression preferentially in the A2/C2 catecholaminergic neurons labelled with the retrograde tracers. These data suggest that the conditioned fear-induced oxytocin cell activity is mediated by the A2 noradrenergic neurons projecting to oxytocin neurons, while the unconditioned fear response is mediated by both A2 and A1 noradrenergic neurons.     

The origin of tyrosine hydroxylase-immunoreactive fibers in the regions of the nucleus basalis magnocellularis of the rat

The simultaneous use of acetylcholinesterase (AChE) histochemistry and tyrosine hydroxylase (T-OH) immunocytochemistry permitted demonstration of the existence of a dense catecholaminergic network surrounding cholinergic neurons within the nucleus basalis magnocellularis (NBM) of the rat. The origin of this catecholaminergic network was investigated by combining T-OH immunocytochemistry with horseradish peroxidase (HRP) retrograde labelling using a slow release gel, unilaterally implanted in the area of the NBM. Retrogradely transported HRP was detected in some of the aminergic cell groups of the substantia nigra (A9) and locus coeruleus (A6). In these areas, approximately 1% of the tyrosine hydroxylase immunoreactive neurons were retrogradely labelled with HRP. In the substantia nigra, dually labelled neurons were found predominantly in the pars lateralis.     

大鼠脑出血致多器官功能障碍综合征模型下丘脑、延髓内脏带与迷走神经的作用

目的 ①探讨脑出血(CH)致多器官功能障碍综合征(MODS)大鼠模型下丘脑、延髓内脏带(MVZ)及迷走神经的作用;②揭示脑源性多器官功能障碍综合征(CMODS)时下丘脑、延髓内脏带及迷走神经的可能调控机制.方法 Wistar大鼠80只用随机数字表法分为:假手术组(10只)、迷走神经切断(SDV)组(10只)、CH组(20只)、脑出血+迷走神经切断(CH+SDV)组(20只)、脑出血+迷走神经刺激(CH+SIV)组(20只).尾状核胶原酶注入法制作大鼠CH模型,偶氮显色鲎试验法定量测定血清内毒素,免疫组织化学法检测下丘脑FOS蛋白及延髓内脏带(MVZ)内乙酰胆碱转移酶(CHAT)的表达.结果 CH+SDV组、CH+SIV组和CH组的肝、肾功能,心肌酶异常程度均重于假手术组和SDV组,CH+SDV组重于CH组,而CH+SIV组明显轻于CH组;CH组血清内毒素含量(EU/m1)与假手术组比较差异有统计学意义(1.03±O.25与0.18±0.03,t=10.61,P<0.01);CH+SDV组下丘脑内FOS蛋白的表达(9.3±2.6)明显多于假手术组(2.5±0.4,t=8.17,P<0.01)和SDV组(2.9±0.5,t=7.64,P<0.01),但明显少于CH组(48.6±18.1,t=6.80,P<0.01),CH+SIV组明显多于CH组(74.3±6.3与48.6±18.1,t=4.23,P<0.01);CH+SDV组的MVZ内CHAT的表达(138.5±11.8)明显多于假手术组(108.2±9.5,t=5.39,P<0.01)和SDV组(110.7±9.9,t=4.83,P<0.01),但明显少于CH组(164.4±12.4,t=3.53,P<0.01),CH+SIV组明显多于CH组(189.4±15.0与164.4±12.4,t=3.14,P<0.05).结论 ①下丘脑与CH致MODS的神经免疫调节有关;②下丘脑-延髓内脏带-迷走神经通路机制可能是CMODS的特性;③迷走神经可能是CH致MODS的双向调节通路.

Abstract：

Objective To investigate the role of the hypothalamus, medullary visceral zone (MVZ)and vagus in cerebral hemorrhage (CH) in multiple organ dysfunction syndrome (MODS) rat model.To reveal the possible regulatory mechanism of the hypothalamus-MVZ-vagus pathway in CMODS.Methods 80 Wistar rats were randomly divided into sham-operative group (n = 10), subdiaphragmatic vagotomy group (SDV, n = 10), CH group (n = 20), CH + SDV group (n = 20) and CH + vagus stimulation group (CH +SIV, n = 20).Rat CH model was made by stereotaxis injection of collagenase in caudate nucleus.Its endotoxin in serum was evaluated with a test kit.The expression of FOS in the hypothalamus and CHAT in the MVZ in the rats were examined using immunohistochemistry methods.Results Abnormality of hepatic and renal function, myocardium enzyme in CH + SDV group, CH + SIV group and CH group were more severe than those in sham-operative group and SDV group; CH + SDV group more severe than CH group.CH +SIV group were much milder than CH group.The serum endotoxin level (EU/ml) in CH group,compared with the sham-operated group, were significantly different (1.03 ± 0.25 and 0.18 ± 0.03respectively, t = 10.61, P <0.01).The expression of FOS in hypothalamus of CH + SDV group (9.3 ±2.6) were significantly higher than those in sham-operative group (2.5 ± 0.4, t = 8.17, P < 0.01) and SDV group (2.9 ±0.5, t =7.64, P <0.01), but significantly lower than those in CH group (48.6 ± 18.1,t =6.80, P<0.01); was higher in CH +SIV group than those in CH group (74.3 ±6.3 and 48.6 ± 18.1,t=4.23, P <0.01).The expression of CHAT in MVZ in CH + SDV group (138.5 ± 11.8) was significantly higher than those in sham-operative group (108.2 ±9.5, t =5.39, P <0.01) and SDV group (110.7 ±9.9, t =4.83, P <0.01), but significantly lower than those in CH group (164.4 ± 12.4, t =3.53, P <0.01); was higher in CH + SIV group than in CH group (189.4 ± 15.0 and 164.4 ± 12.4, t =3.14, P < 0.05).Conclusion Hypothalamus may be related to the nerve immunity adjustment in MODS after cerebral hemorrhage.The hypothalamus-MVZ-vagus pathway may be distinctive in CMODS.Vagus nerve may be a Bi-directional Channel of the rats following cerebral hemorrhage and MODS.     

Rhythmicity of β-endorphinergic neuronal activity in the mediobasal hypothalamus during pregnancy in the rat

During the first half of gestation in the rat, prolactin (PRL) from the anterior pituitary gland exerts its luteotropic function on the ovary to stimulate progesterone secretion. During this period, β-endorphin stimulates PRL secretion by regulation of dopaminergic neurons in the hypothalamus. During the second half, placental lactogens (PLs) take the place of PRL in maintenance of pregnancy, and initiate a negative feedback to suppress PRL secretion. However, the effect of PLs on β-endorphinergic neurons is not known. The aim of this study was to examine the possibility that PLs suppress PRL secretion by inhibiting β-endorphinergic neuronal activity. To accomplish this aim, we examined the changes in the neuronal activity of β-endorphinergic neurons in the mediobasal hypothalamus, as measured by Fos immunoreactivity, after manipulating the levels of PRL and PLs during pregnancy. On day 4 of pregnancy, animals received either Rcho-1 cells in the lateral ventricle that secrete PLs or HRP-1 cells as controls. In a separate experiment on day 12, hysterectomy was performed to remove the intrinsic source of PLs. These rats received Rcho-1 cells, HRP-1 cells, or nothing. Intracerebroventricular (i.c.v.) injection of Rcho-1 into hysterectomized rats was done to examine the effect of PL replacement. Sham-hysterectomy was also performed as a control. Animals were sacrificed 2 days after each treatment at 0200 h, 1400 h, and 1800 h. Brains were used for dual immunocytochemistry of Fos/β-endorphin. The neuronal activity of β-endorphinergic neurons of HRP-1 i.c.v. injected animals showed a daily rhythm, with high levels at 0200 h and 1800 h, and a low level at 1400 h. These animals also exhibited two surges of PRL secretion on day 6 of pregnancy. This rhythmicity of β-endorphinergic neurons was also observed in Rcho-1 i.c.v. injected animals, which showed very low and unchanging PRL levels. However, the magnitude of neuronal activity was reduced. On day 14 of pregnancy, all four experimental groups showed diurnal rhythms of β-endorphinergic neurons. This rhythmicity occurred even though PRL was elevated at all three time points in the hysterectomized rats and very low in the Rcho-1 i.c.v. injected hysterectomized and sham-hysterectomized rats. Our results demonstrate that there is a diurnal rhythm of β-endorphinergic neuronal activity in the mediobasal hypothalamus during pregnancy in the rat. PLs might reduce the neuronal activity of β-endorphinergic neurons, but only during the first half of pregnancy, partially explaining the suppression of PRL surges.     

蛛网膜下腔出血致多器官功能障碍综合征大鼠FOS蛋白的表达研究

目的通过研究大鼠蛛网膜下腔出血（SAH）致多器官功能障碍综合征（MODs）延髓内脏带（MVZ）FOS蛋白的表达,探讨MVZ在MODs中的作用。方法Willis环注血法建立大鼠sAH致MODS模型,48只Wistar大鼠随机分为正常时照组、假手术组、SAH后6个亚组（4,12,24,36,48,72h组）,依据全身炎症反应综合征（SIRS）和MODS的诊断标准判断sIRS和MODS的发生率,应用免疫组织化学法检测MVZ内FOS蛋白的表达。结果（1）SAH后SIRS发生率为100％,MODS发生率为69．4％;（2）大鼠SAH后12hMVZ内FOS蛋白表达升高,24～36h达到高峰,密集分布于MVZ背内侧部的孤束核、最后区、迷走神经背核和外侧网状核,与正常对照组和假手术组比较有显著性差异（P〈0．01）,48h后表达减少;（3）大鼠SAH后各时相点各脏器存在不同程度的炎性损害,以24～36h病理变化最显著,与FOS蛋白的表达峰值一致。结论（1）Willis环注血法可成功建立大鼠SAH致MODS的动物模型;（2）MVZ参与了SAH后周围脏器功能的调控,是SAH致MODS的直接调控中枢之一。     

Gastric electrical stimulation increases ghrelin production and inhibits catecholaminergic brainstem neurons in rats

Gastric electrical stimulation (GES) is a new therapeutic option for functional dyspepsia and gastroparesis. In addition to ameliorating nausea and vomiting, GES results in improved appetite which is not always associated with accelerated gastric emptying. To explore the central and peripheral factors underlying GES‐associated improvement of appetite we developed a GES model in anaesthetized Wistar rats. During laparotomy, two electrodes were implanted into the stomach and high‐frequency low‐energy GES (14 Hz, 5 mA) was applied. The effects of 1 h GES were compared with sham stimulation. After GES, c‐Fos expression was increased in the mucosal and submucosal layers of the stimulated area (174%). In the stomach, GES increased ghrelin mRNA (178%) and doubled the number of ghrelin‐positive cells, resulting in elevated plasma levels of ghrelin (2.3 ± 0.2 vs. 1.6 ± 0.2 ng/mL). In the arcuate nucleus of the hypothalamus, GES increased c‐Fos (277%) and agouti‐related protein (AgRP) mRNA expression (135%). GES reduced the number of c‐Fos‐positive cells throughout the nucleus of the solitary tract (between 93 and 75% from rostral to caudal levels) including catecholaminergic neurons (81% at caudal level). Gastric emptying, plasma glucose and heart rate variability were not affected by GES. This study shows that GES may improve appetite via stimulation of main orexigenic pathways, including ghrelin production in the stomach and AgRP in the hypothalamus, as well as by reducing the activity of catecholaminergic brainstem neurons.     

Subdiaphragmatic vagotomy increases the sensitivity of lumbar Aδ primary afferent neurons along with voltage-dependent potassium channels in rats

Subdiaphragmatic vagal dysfunction causes chronic pain. To verify whether this chronic pain is accompanied by enhanced peripheral nociceptive sensitivity, we evaluated primary afferent neuronal excitability in subdiaphragmatic vagotomized (SDV) rats. SDV rats showed a decrease in the electrical stimuli-induced hind limb-flexion threshold at 250 Hz, but showed no similar effect at 5 or 2000 Hz, which indicated that lumbar primary afferent Aδ sensitivity was enhanced in SDV rats. The whole-cell patch-clamp technique also revealed the hyper-excitability of acutely dissociated medium-sized lumbar dorsal root ganglion (DRG) neurons isolated from SDV rats. The contribution of changes in voltage-dependent potassium (Kv) channels was assessed, and transient A-type K(+) (I(A) ) current density was apparently decreased. Moreover, Kv4.3 immunoreactivity in medium-sized DRG neurons was significantly reduced in SDV rats compared to sham. These results indicate that SDV causes hyper-excitability of lumbar primary Aδ afferent neurons, which may be induced along with suppressing I(A) currents via the decreased expression of Kv4.3. Thus, peripheral Aδ neuroplasticity may contribute to the chronic lower limb pain caused by SDV.     

Activation of brainstem catecholaminergic neurons during voluntary diving in rats

Underwater submergence produces a complex autonomic response that includes apnea, a parasympathetically-mediated bradycardia, and a sympathetically-mediated increase in total peripheral resistance (TPR). The present study was designed to identify brainstem catecholaminergic neurons that may be involved in producing the increased TPR during underwater submergence. Twelve male Sprague-Dawley rats were trained to voluntarily dive 5 m through an underwater maze. On the day of the experiment the rats were randomly separated into a Diving group that repetitively dived underwater, a Swimming group that repetitively swam on the surface of the water, and a Control group that remained in their cages. After the experiment the brainstems of the rats were immunohistologically processed for Fos as an indicator of neuronal activation, and for tyrosine hydroxylase (TH) as an indentifier of catecholaminergic neurons. Neurons labeled with both Fos and TH identified activated catecholaminergic neurons. In Diving rats there was increased Fos+TH labeling in A1, C1, A2, A5, and sub-coeruleus, as well as globosa neurons in the lateral A7 region compared with Control rats, and in A1, C1 and A5 compared with Swimming rats. In Swimming rats Fos+TH labeling was significantly increased in caudal A1, A5, sub-coeruleus and globosa neurons compared with Control rats. These data suggest that selective groups of catecholaminergic neurons within the brainstem are activated by voluntary underwater submergence, and some probably contribute to the sympathetically-mediated increase in vascular tone during diving.