大鼠坐骨神经和盆神经初级传入纤维向腰骶髓后连合核的投射--HRP和BSI-B4-HRP跨越神经节追踪研究

目的　观察大鼠坐骨神经和盆神经初级传入纤维在腰骶段脊髓后连合核内的分布。　方法　应用HRP和BSI B4 HRP跨越神经节追踪技术。　结果　将HRP注射到坐骨神经后 ,有一定数量的HRP标记纤维出现于后连合核内 ,而BSI B4 HRP标记的坐骨神经初级传入纤维全部终止于后角浅层 (主要在Ⅱ层 ) ,后连合核内未见任何阳性标记。HRP标记的后根节神经元大、中、小均有 ,其平均直径为 33 2 5± 14 18μm ,而BSI B4 HRP标记的细胞以小型为主 ,其平均直径为 17 5 9± 4 80 μm。HRP和BSI B4 HRP标记的盆神经初级传入纤维在腰骶段脊髓内的分布相似 ,且均向后连合核投射 ,但BSI B4 HRP注入例的标记量明显少于HRP实验组。BSI B4 HRP标记的后根节神经元的数量也明显少于HRP实验组 ,但两者均以直径在 10～ 2 0 μm的小型细胞为主。 　结论　终止于后连合核的坐骨神经初级传入纤维可能为粗纤维 ,而盆神经则含有细纤维。这种躯体和内脏神经在后连合核内的不同终止形式可能与针刺镇痛的机制密切相关     

大鼠膈神经传出和传入神经成分在脊髓内的分布

采用HRP追踪法,对成年大鼠膈神经运动纤维的起源和初级传入神经元胞体的位置及在其中枢突脊髓的投射进行了观察。结果表明,膈神经运动纤维在脊髓标记(膈核)主要分布在注射侧C_3-C_5脊髓节段的前角。隔神经感觉纤维初级传入标记的神经元见于注射侧C_3-C_6后根节,其中C_4标记的细胞数最多。     

跨越神经节追踪中HRP运输规律的探讨

用家兔37只,向左侧小腿三头肌神经(肌神经)和右侧腓肠皮神经(皮神经)注入HRP溶液,分别存活5 1/2、6、7、10、12、15、18、21、24小时及2、3、5、14、16、18、19天。TMB反应。在光镜下观察后根节细胞及脊髓内初级传入纤维及终末的标记状态及消长过程,取得了下列几点规律性认识。 1.后根节从6小时出现标记细胞,到18—19天标记完全消退。全程经历了标记由淡变浓又变淡终于消失的过程,大体上可分为增长期、高峰期和消退期三个阶段。小细胞标记出现早(6h)、消退也早(5天时巳有很多消退);大细胞标记出现迟、消退也慢。反映着神经元摄取的HRP量的多少决定着细胞标记程度和标记存续时间的长短。 2.髓内的纤维和终末从12小时出现标记,5天全部消褪,也是细纤维先出现、先消退,粗纤维后出现、后消退。 3.各种不同类型的后根节细胞的标记消退形式也不相同。小细胞或中小细胞胞体内的酶颗粒均匀地变淡而全面消退;中型细胞则靠近胞膜处先消退,核周围的酶颗粒暂时保留;大及特大型细胞胞体内出现空泡,有的空泡融合形成大的空泡,核也随之偏位,显示为退行性变。 4.在跨节传递过程中,逆行运输的速度快于顺行运输。     

骶髓后连合核接受盆内脏伤害性信息传入的形态学证明

为阐明投射至骶髓后连合核的盆内脏初级传入中是否含有传递伤害性信息成分，本研究综合运用特异性标记初级传入Ｃ纤维的ＢＳＩ－Ｂ４－ＨＲＰ跨节追踪技术，神经干局部涂抹Ｃ纤维毒素ＣａＰｓａｉｃｉｎ并结合ＳＰ免疫组化方法，研究了猫投射至骶髓后连合核的盆神经初级传入纤维中是否含有传递伤害性刺激的成分；同时观察了秋水仙素处理的骶２后根节内ＢＳＩ－Ｂ４标记的初级传入神经元与ＳＰ免疫阳性神经元的关系。结果如下：（１）向盆神经注入ＢＳＩ－Ｂ４－ＨＲＰ，骶１～３后根节内出现平均直径３４μｍ的标记细胞，后连合核内出现密集的标记终末，电镜下证明通过Ｌｉｓｓａｕｅｒ氏束进入脊髓内的标记纤维均为无髓纤维；（２）对盆神经进行局部Ｃａｐｓａｉｃｉｎ处理，引起后连合核内的ＳＰ免疫阳性纤维和终末明显减少；（３）骶２后根节内ＢＳＩ－Ｂ４－ＦＩＴＣ标记细胞有１７％同时呈ＳＰ免疫阳性；（４）骶２后根节内ＢＳＩ－Ｂ４－ＨＲＰ标记的盆内脏初级传人神经元的３９％同时呈ＳＰ免疫阳性。本研究结果在形态学上证实了骶髓后连合核接受盆腔内脏伤害性信息的传入，它可能是中继和整合盆内脏伤害性信息的低级中枢。     

大鼠胸腺传入纤维在中枢内的跨节投射——CB-HRP法

本文将CB-HRP注入大鼠胸腺,研究了标记细胞在感觉神经节的分布及初级感觉终末在脊髓、脑干中的投射。结果表明:1.在双侧迷走神经结状节中有标记细胞;2.在C_1-T_5段双侧脊神经节中有标记细胞,推测这些标记细胞可能随交感神经传入,其中C_3-C_4段的投射可能随膈神经传入;3.脊髓中的标记终末,见于C_1-C_8节段,主要分布在Ⅰ层内;4.脑干中的标记终末,主要见于双侧孤束核的全长及连合核中。在疑核标记的节前神经元附近有的也可见终末分布,提示胸腺的一级传入与传出神经元间可能存在直接联系。     

躯体和内脏SP能初级传入神经元的定位研究

本文在后根节和脊髓两个水平对躯体(胫神经)和内脏(膀胱)初级传入系统进行同时定性、定位的研究。对后根节选用两种较敏感的双标技术(HRP结合PAP,Biotin-WGA结合免疫荧光)。结果表明,在后根节中被标记的胫神经和膀胱的初级传入神经元均有一部分同时显示SP样阳性反应。另外在分别切断盆内脏神经和胫神经的两组动物模型上进行脊髓的SP样免疫反应,通过两侧对比,观察到术侧灰质在相当于被切断神经的传入经路及终末区出现SP样反应的“脱落”现象,从而反证出躯体和内脏初级传入纤维中的SP能成分在中枢内的定位分布。本文首次提供了内脏初级传入在中枢内定位与定性相结合的研究结果。     

家兔子宫初级感觉神经元在脊神经节的节段性分布——HRP逆轴突追踪法

本实验用HRP法对家兔子宫初级感觉神经元在脊神经节的节段性分布规律进行了研究。酶标细胞出现在T_9～Co_1脊神经节范围内。较集中于L_(2～5)和S_(3、4)脊神经节,尤以L_4和S_3节最多。而L_(6、7),S_1节很少。胸腰段与骶尾段酶标细胞数的比约为5:1。兔子宫以源自颈部标记神经元数量较多。源自子宫体中部的标记神经元分布较少,上、卜两部标记的神经的数量相近。子宫各部感觉的传入节段一致。子宫的初级感觉神经元,以小细胞为主,中细胞次之,大细胞最少。初级感觉,均传入同侧脊神经节中,左右未见交叉现象。     

大鼠三叉神经节细胞向三叉神经脊束核各亚核的分枝投射

本文应用荧光素双标记与免疫荧光结合方法研究了单个含ＳＰ或ＣＧＲＰ的三叉神经节细胞向三叉神经脊束核的尾侧亚核、极间亚核和吻侧亚核的分枝投射。将ＤｉａｍｉｄｉｎｏＹｅｌｌｏｗ（ＤＹ）注入尾侧亚核，ＦａｓｔＢｌｕｅ（ＦＢ）分别注入吻侧亚核或极间亚核，发现ＤＹ／ＦＢ双标细胞占同侧三叉神经节内标记细胞总数的１３．２％（ＦＢ注入吻侧亚核例）和２．３％（ＦＢ注入极间亚核例）；双标细胞中含ＳＰ者分别为７４．４％和６９．６％；含ＣＧＲＰ者分别为７２．１％和６４．３％。将ＤＹ注入极间亚核，ＦＢ注入吻侧亚核时，ＤＹ／ＦＢ双标细胞占同侧三叉神经节标记细胞总数的１．６％，双标细胞中有６７．９％为ＳＰ阳性，７３．９％为ＣＧＲＰ阳性。多数的双标且呈ＳＰ或ＣＧＲＰ阳性的三叉神经节细胞直径约为２５～５０μｍ，为中、小型节神经元，而直径大于５０μｍ的大型细胞较少见。以上结果提示，三叉神经初级传入纤维进入脑干后的下降支分枝投射向三叉神经脊束核的各亚核，它们可能与面口部的痛信息传递有关。ＳＰ和ＣＧＲＰ是这些分支投射神经元的重要神经活性物质。     

孤束核内儿茶酚胺能神经元汇聚内脏传入与口面部躯体伤害性信息并向臂旁核投射

为了研究内脏初级传入与口面部深层组织躯体伤害性信息是否汇聚于孤束核(NTS)中向臂旁核(PBN)投射的儿茶酚胺(CA)能神经元。本实验将SD大鼠随机分为两组。第一组动物以生物素化的葡聚糖胺(BDA)注入颈部迷走神经主干跨节追踪,四甲基罗达明(TMR)注入臂旁外侧核(LPB)逆行追踪,福尔马林刺激咬肌并行FOS的免疫荧光组织化学染色;第二组动物以BDA注入颈部迷走神经主干跨节追踪,福尔马林刺激咬肌并行FOS和酪氨酸羟化酶(TH)染色。在激光扫描共聚焦显微镜下对两组大鼠NTS内的标记细胞和纤维进行了观察。两组实验中,主要在NTS的内侧、中间内侧和连合亚核内观察到重叠分布的TMR、FOS或TH、FOS单标神经元。其中部分TMR逆行标记的或TH阳性神经元呈FOS阳性并与BDA跨节标记的纤维和终末形成紧密接触。提示大鼠NTS内的CA能神经元可能汇聚了内脏初级传入和口面部深层组织的躯体伤害性刺激信息并向LPB投射。     

路过猫星状神经节的传入神经元的节段性分布——HRP法的研究

本实验将33只猫分为两组,第一组在左或右星状神经节内注射HRP后,标记细胞分布在注射同侧的C_4～T_(?)脊神经节,大多数标记细胞集中分布在T_(1～5)脊神经节。左、右侧标记细胞的节段性分布和分布模式上没有明显差别。各节段的标记细胞大多数为50μm以下的中、小型细胞。另一组切断椎神经后将HRP注入星状神经节,标记细胞仅分布在T_(1～9)脊神经节,证明椎神经是C_(4～8)脊神经节细胞发出传入纤维路经星状神经节的联系通路。     

山羊迷走神经感觉纤维的来源——HRP法研究

为了探讨山羊迷走神经感觉纤维的来源,本文将HRP注入颈迷走神经干后,在结状节出现大量密集的标记细胞,颈静脉节中也有较多的细胞被标记,但其密度和数量远不如结状节。在颈1—8和胸1—3的背根节中出现一定数量的标记细胞。此外,少量的标记细胞见于迷走神经干的纤维束中,这些标记细胞的形态与结状节的基本相同。     

The origin of sensory innervation of the peritoneum in the rat

The distribution of sensory neurons innervating the peritoneum was studied using axonal transport of fluoro-gold. The tracer was injected into parietal peritoneum, diaphragm, mesentery, mesocolon, visceral peritoneum covering the stomach, small intestine, colon, liver, spleen, kidney, urinary bladder or uterus. After ten days of survival bilateral dorsal root ganglia from C2 to S6, and the nodose ganglia were dissected. The cryostat sections of these ganglia were mounted on glass slides and observed with a fluorescence microscope. In cases where the tracer was placed on the peritoneum covering the abdominal wall, labeled neurons were observed only in the ipsilateral dorsal root ganglia. A small number of neurons in nodose and cervical dorsal root ganglia of both sides were labeled after placing the tracer on the central part of the diaphragm. When fluoro-gold was applied to the peripheral part of the diaphragm, nodose ganglion was negative, and dorsal root ganglia from T6 to T12 were positive. Many neurons in the nodose ganglia in addition to somata in the dorsal root ganglia from T4 to T13 were labeled when the tracer was placed on the peritoneum lining the stomach, small intestine or caecum. After applying the tracer onto the colon, labeled neurons were observed in the dorsal root ganglia from T13 to L2 and L5 to S1. Ganglion cells in the nodose and dorsal root ganglia from T5 to T13 were positive when fluoro-gold was placed on the mesentery. No labeled neurons were observed in any ganglia when the tracer was applied to the peritoneum covering the spleen, kidney, uterus, urinary bladder and liver. These results suggest that most of the parietal peritoneum receives sensory nerves from dorsal root ganglia and the visceral peritoneum from both spinal nerves and the vagus nerve.     

Distribution of the cells of primary afferent fibers to the cat auricle in relation to the innervated region.

The origin and peripheral distribution of the primary afferent fibers to the cat auricle were studied by the horseradish peroxidase (HRP) method. Following HRP injection into the whole region of the auricle, HRP-labeled cells were found ipsilaterally in the trigeminal ganglion, geniculate ganglion, superior ganglion of the vagus nerve and in the C1 to C4 spinal ganglia. In addition to the ganglia of cranial and spinal nerves, labeled cells were also observed in the facial nerve trunk and in the dorso-lateral portion of the superior cervical ganglion. In the case of HRP injection into the central region of the auricle, labeled cells were principally observed in the ganglia of cranial nerves and to a lesser degree in the spinal ganglia. On the contrary, in the case of HRP injection into the peripheral region of the auricle, labeled cells were principally observed in the spinal ganglia, although some were seen in the ganglia of cranial nerves. This study suggests that the cutaneous innervation of the auricle is supplied by both the cranial and spinal nerves, and that the central region of the auricle is strongly innervated by the cranial nerves and the peripheral region of the auricle is strongly innervated by the spinal nerves.     

Interneuronal signalling is involved in induction of collateral sprouting of nociceptive axons

Collateral sprouting of cutaneous nociceptive axons into the adjacent denervated skin critically depends on the nerve growth factor, presumably originating from the degenerated neural pathways and denervated skin. We hypothesised that the degenerated neural pathways are necessary, but not sufficient, to induce collateral sprouting of nociceptive axons, and, in addition, that the interaction between the injured and non-injured neurones within a dorsal root ganglion can trigger sprouting of nociceptive axons also in the absence of the denervated skin. End-to-side nerve anastomosis, made in female Wistar rats by suturing the end of an excised peroneal nerve segment to the side of the intact sural nerve, was used as a model for sprouting which allowed us to study the putative induction mechanisms separately. If the nerves adjacent to the sural nerve were transected concomitantly with the coaptation of the end-to-side anastomosis, robust nociceptive axon sprouting into the anastomosed nerve segment was observed by the nerve pinch test and counting of myelinated axons. Collateral sprouting did not occur, however, either if the cells in the anastomosed nerve segment were killed by freezing and thawing, or if the adjacent nerves had not been injured. However, if the ipsilateral dorsal cutaneous nerves, having their neurones in the same dorsal root ganglia as the sural nerve, were transected, but no other nerves were injured, then the sural nerve axons sprouted in abundance through the anastomosis even in the absence of denervated skin around the sural nerve terminals.From these results we suggest that cells (probably proliferating Schwann cells) in the degenerated neural pathways are necessary but not sufficient to induce collateral sprouting of nociceptive axons, and that interactions between the injured and non-injured neurones within the dorsal root ganglion (i.e. direct or indirect interneuronal signalling) are important in this regard.     

Survival and regeneration of cutaneous and muscular afferent neurons after peripheral nerve injury in adult rats

Peripheral nerve injury induces the retrograde degeneration of dorsal root ganglion (DRG) cells, which affects predominantly the small-diameter cutaneous afferent neurons. This study compares the time-course of retrograde cell death in cutaneous and muscular DRG cells after peripheral nerve transection as well as neuronal survival and axonal regeneration after primary repair or nerve grafting. For comparison, spinal motoneurons were also included in the study. Sural and medial gastrocnemius DRG neurons were retrogradely labeled with the fluorescent tracers Fast Blue (FB) or Fluoro-Gold (FG) from the homonymous transected nerves. Survival of labeled sural and gastrocnemius DRG cells was assessed at 3 days and 1–24 weeks after axotomy. To evaluate axonal regeneration, the sciatic nerve was transected proximally at 1 week after FB-labeling of the sural and medial gastrocnemius nerves and immediately reconstructed using primary repair or autologous nerve grafting. Twelve weeks later, the fluorescent tracer Fluoro-Ruby (FR) was applied 10 mm distal to the sciatic lesion in order to double-label sural and gastrocnemius neurons that had regenerated across the repair site. Counts of labeled gastrocnemius DRG neurons did not reveal any significant retrograde cell death after nerve transection. In contrast, sural axotomy induced a delayed loss of sural DRG cells, which amounted to 22% at 4 weeks and 43–48% at 8–24 weeks postoperatively. Proximal transection of the sciatic nerve at 1 week after injury to the sural or gastrocnemius nerves neither further increased retrograde DRG degeneration, nor did it affect survival of sural or gastrocnemius motoneurons. Primary repair or peripheral nerve grafting supported regeneration of 53–60% of the spinal motoneurons and 47–49% of the muscular DRG neurons at 13 weeks postoperatively. In the cutaneous DRG neurons, primary repair or peripheral nerve grafting increased survival by 19–30% and promoted regeneration of 46–66% of the cells. The present results suggest that cutaneous DRG neurons are more sensitive to peripheral nerve injury than muscular DRG cells, but that their regenerative capacity does not differ from that of the latter cells. However, the retrograde loss of cutaneous DRG cells taking place despite immediate nerve repair would still limit the recovery of cutaneous sensory functions.     

Some nerve endings in the rat pelvic paracervical autonomic ganglia and varicosities in the uterus contain calcitonin gene-related peptide and originate from dorsal root ganglia

The pelvic paracervical autonomic ganglia of female rats were studied for a subpopulation of nerve endings that could be derived from sensory nerve fibers. Immunohistochemical staining using an antiserum against the synaptic-terminal protein synapsin I was used to identify terminal boutons, while an antiserum against the neuropeptide calcitonin gene-related peptide was used to reveal a subpopulation of sensory nerve fibers. The uterine cervix was also examined for the existence of calcitonin gene-related peptide and synapsin I immunoreactivity in nerve fiber varicosities. In addition, the location of nerve endings in the paracervical ganglion was compared to that in the superior cervical ganglion. Synapsin I immunoreactivity was present in the paracervical ganglion in abundant boutons around neuron somata and in the cervix in varicose nerve fibers of the myometrium, vasculature and epithelium. Double labeling immunocytochemistry revealed calcitonin gene-related peptide-like immunoreactivity in subpopulations of synapsin I-immunoreactive endings in ganglia and nerve varicosities in the cervix. Injection of a retrograde axonal tracer, fluorogold, into the paracervical ganglion produced labeled neurons in dorsal root ganglia and spinal cord; however, fluorogold-labeled neurons containing calcitonin gene-related peptide immunoreactivity were visualized only in dorsal root ganglia. Injections of fluorogold into the uterine cervix produced labeled neurons in the paracervical ganglion and dorsal root ganglia; however, only those in dorsal root ganglia contained immunoreactivity for calcitonin gene-related peptide. These results suggest that immunoreactivity for calcitonin gene-related peptide is present in a subpopulation of nerve endings in the paracervical ganglion and not merely in fibers of passage. The nerve endings in the ganglion and varicosities in the uterine cervix originate from sensory neurons in dorsal root ganglia. The arrangement of endings in the ganglia could play a role in sensory/autonomic interactions for modulation of visceral activity.     

Morphometrical study of the cat thoracic dorsal root ganglion cells in relation to muscular and cutaneous afferent innervation

The sizes of neuronal somata in cat dorsal root ganglia were determined at the different thoracic segmental levels (T1-T13). The intersegmental variations in the average value and class distribution of diameters were analysed. The maximal and minimal average mean cell diameters were 51.1 and 43.3 microns at the T1 and T2 levels, respectively. Caudally, this value gradually increased from T2 to T8 (47.8 microns) and thereafter decreased progressively to T12 (44.7 microns). At T1, large cells (greater than 50 microns in diameter) were 3.3-fold in excess compared to small ones (less than 35 microns in diameter). The proportion of large to small cells strongly decreased to a 0.9 ratio from T1 to T2, then increased again from T2 (0.9) to T8 (2.3). The size distributions of the overall cell populations were compared to those of neurones supplying muscular targets via the external intercostal nerves or cutaneous targets via the lateral branch of the internal intercostal nerves, identified following the retrograde transport of horseradish peroxidase. The size distribution of cells serving cutaneous nerves was similar to that exhibited by the overall population of ganglion cells. In contrast, the size distributions of cells giving rise to muscle afferents tended towards smaller values. In the thoracic dorsal root ganglia, the cell body sizes of the muscular primary afferents were close to those previously reported for the visceral primary afferents.     

胆囊传入神经的节段性分布——CT-HRP法研究

将CT—HRP注入胆囊壁游离面,取脊神经节、迷走神经结状节作TMB法反应。结果是(1)胸2—13和腰1节段的脊神经节内有大量的标记细胞,右侧者数量占优势,左右胸7—10节标记细胞数量为高峰节段。(2)仅在右颈5—7脊神经节中发现不同数量的标记细胞,出现率约占实验动物的1/3,切断右侧膈神经后在颈部脊髓神经节中不出现标记细胞。(3)双侧迷走神经结状节中均发现标记细胞,左右侧在数量上无明显差异。脊神经节或结状节的标记细胞均以中小型为主,也有少最大型细胞。