Morphological Changes of the Sensory Neurons in the Peripheral Neuropathy of Rat Tibial Nerve Using WGA-HRP Tracing Method / 체질인류학회지

Neuropathy is a general term referring to disorders of nerves, and produces when the nerves are damaged. It is characterized by spontaneous pain, allodynia and hyperalgesia. The purpose of present study is to observe the number of WGA-HRP (wheat germ agglutinin-horseradish peroxidase) labelded sensory neurons of DRG (dorsal root ganglia), and distributions according to cell size of sensory neuron in tibial nerve ligation model (NLM). The tibial nerve ligation was performed with 3-0 silk by the application of three tight ligatures at the mid-thigh level. In the neuropathy model of rat tibial nerve ligation, morphological changes of sensory neurons in DRG were observed using WGA-HRP. Rats of NLM showed the neuropathic behaviors. Rats were shown guarding affected limb and limping. Their toes and ankle joint of operated limb were hyperflexed. Under light microscopy, tibial nerve showed degeneration of axons in NLM. In control and NLM, labeled sensory neurons of tibial nerve distributed L4 and L5 DRG. In control group, the labeled sensory neurons were round or oval in shape. They were large and small cells, and mixed pattern. Total number of labeled sensory neurons in NLM decreased significantly from control group. The number of labeled sensory neurons in L4 and L5 DRG decreased significantly from control group. Labeled large and small cells decreased significantly from control group. Present study may serve as the basic information about the changes of DRG sensory neurons in NLM.

Brainstem origin of duodenal vagal preganglionic parasympathetic neurons. A WGA-HRP study in the ferret (Mustela Putorius Furo), a human model

The projections of vagal brainstem neurons to the duodenal segment of the gastrointestinal tract were studied in the ferret using the WGA-HRP neurohistochemical technique. Fourteen adult ferrets with weights ranging from 800 gm to 1500 gm were used for the study. The muscular wall of the duodenum of six ferrets was injected with 0.1 ml of 5 WGA-HRP in 0.5 M sodium chloride. The eight remaining ferrets were used as controls. Two of these had injections of 0.1 ml normal saline into the muscular wall of the duodenum. The second set of two ferrets was injected with 0.1 ml of 5 WGA-HRP in buffer after bilateral truncal vagotomy. The third set of two ferrets received intraperitoneal injection of 0.1 ml of 5 WGA-HRP while, in the last set, the tracer was injected into the hepatic portal vein. Following the injections, the ferrets were allowed to survive for 48-72 hours after which each ferret was perfused transcardially first with normal saline followed by a fixative containing 1 paraformaldehyde and 1.25 glutaraldehyde in 0.1 M phosphate buffer, pH 7.4 at room temperature and finally with 10 buffered sucrose at 4 degrees C. Transverse serial frozen sections of the brainstem were then taken and processed for WGA-HRP neurohistochemistry and were analyzed under light and dark-field illuminations. The analyses of the sections taken from the six ferrets injected with WGA-HRP revealed neurons labelled with the tracer in the dorsal motor nucleus of the vagus nerve (DMNV). Sections taken from the control ferrets did not reveal any WGA-HRP labelled neurons in the brainstem

A Study on the Spinoreticulocerebellar Tract in Chickens

The spicoreticulocerebellar (SRC) tract is an indirect spinocerebellar tract formed by the reticular formation (RF), which is connected to the cerebellum and spinal cord. The RF receives ascending fibers to both the spinal enlargement and sends descending fibers to the cerebellum. This study demonstrated that the connectivity of the neurons in the RF is concerned to the cerebellum and spinal cord using the anterograde projection with biotinylated dextran amine (BDA) and retrograde labeling with wheat germ agglutinin-horseradish peroxidase (WGA-HRP). Until now, a preliminary study in mammals has dealt with the afferent and efferent pathways in separating groups of neurons in the RF. There are only few reports on chickens. This study examined the SRC tract in chickens. Following bilateral injections we injected BDA into chicken spinal cord (lumbosacral enlargement) and WGA-HRP into the cerebellum. Both of single- and double-labeled cells were found within the RF. The spinoreticular axons were mainly distributed from the potomedullary junction to the rostral medulla in the rostro-caudally RF levels, for example, nucleus of reticularis (n. r.) pontis oralis, locus coeruleus, n. r. pontis caudalis, n. r. pars gigantocellularis, n. r. gigantocellularis and n. r. parvocellualris. Reticulocerebellar labeling by the WGA- HRP was found in the same place as well as that of the BDA-projection. We observed that the proportion and location of double labeling cells in the chicken were almost similar in each level, comparing to the rodents. These results suggest that the reticular formation is strongly related to the spicoreticulocerebellar tract in chickens.

Efeito inflamatório local induzido pelas lectinas PHA, WGA e jacalina / Local inflammatory effects of PHA, WGA and jacalin lectins

Muitas das atividades inflamatórias atribuídas às lectinas são decorrentes da atividade quimiotáxica, da secreção de citocinas pelos leucócitos ativados e da estimulação policlonal de linfócitos. Este trabalho teve como objetivo avaliar o efeito inflamatório local induzido pelas lectinas PHA, WGA e jacalina. Para tanto, injeções intradérmicas das lectinas nas concentrações de 25 µg/mL, para PHA e jacalina, e 100 µg/mL, para WGA, foram realizadas no dorso de ratos e avaliações macroscópicas e histológicas foram realizadas. Nas avaliações macroscópicas, os diâmetros das pápulas formadas nos locais das injeções foram medidos diariamente. As análises histológicas foram realizadas em cortes corados com hematoxilina-eosina, nos períodos de 24, 48, 72 horas e no 5º e 7º dias. O padrão macroscópico de reação foi semelhante para PHA e jacalina e menor para WGA. A análise histológica evidenciou reação inflamatória bem localizada e forte nas primeiras 24 horas, com predomínio de células mononucleares na inflamação provocada por PHA e jacalina. Após este período foi evidente a diminuição da inflamação provocada por WGA, porém, para PHA e jacalina a reação inflamatória nas próximas 48 horas foi maior e, a partir daí, diminuiu com o tempo, embora, em todos os períodos analisados, foi ligeiramente maior para PHA

Neural Pathway Innervating Epididymis of Rats by Pseudorabies virus (PRV-Ba-Gal) and WGA-HRP / 대한해부학회지

This experimental studies was to investigate the location of PNS and CNS labeled neurons following injection of 2% WGA-HRP and pseudorabies virus (PRV), beta-galactosidase inserted Bartha strain, into the epididymis of rats. After survival times 4~5 days following injection of 2% WGA-HRP and PRV-Ba-Gal, the rats were perfused, and their brain, spinal cord, sympathetic ganglia and spinal ganglia were frozen sectioned (30 mm). These sections were stained by HRP histochemical and beta-galactosidase histochemical staining methods, and observed with light microscope. The results were as follows : 1. The WGA-HRP labeled sympathetic ganglia projecting to the epididymis were observed in pelvic ganglion and L1-6 lumbar sympathetic ganglia. 2. The WGA-HRP labeled spinal ganglia projecting to the epididymis were observed in L1-6 spinal ganglia. 3. The beta-galactosidase labeled neurons projecting to the epididymis were observed in lamina VII of cervical segments. In thoracic segments, beta-galactosidase labeled neurons were observed in dorsomedial part of lamina I, II and III. Dense labeled neurons were observed in intermediolateral n. and dorsal commissural n.. In lumbar segment, labeled neurons were observed in lamina III, IV, V, dorsal commisural n. and superficial dorsal horn. 4. In the medulla oblongata, beta-galactosidase labeled neurons projecting to the epididymis were observed in the trigeminal spinal n., A1 noradrenalin cells/C1 adrenalin cells/caudoventrolateral reticular n., rostroventrolateral reticular n., area postrema, n. tractus solitarius, raphe obscurus n., raphe pallidus n., raphe magnus n., parapyra-midal n., lateral reticular n. and lateral paragigantocellular reticular n.. 5. In the pons, labeled neurons were observed in Kolliker-Fuse n., locus coeruleus, subcoeruleus n. and A5 noradrenalin cells. 6. In midbrain, labeled neurons were observed in periaqueductal gray substance, retrorubral n., substantia nigra and dorsal raphe n.. 7. In the diencephalon, labeled neurons were observed in paraventricular hypothalamic n., lateral hypothalamic nucleus., medial preoptic n. and retrochiasmatic n.. These results suggest that WGA-HRP labeled neurons of the spinal cord projecting to the rat epididymis might be the first-order neurons related to the viscero-somatic sensory and sympathetic postganglionic neurons, and beta-galactosidase labeled neurons of the brain and spinal cord may be the second and third-order neurons response to the movement of vascular smooth muscle in epididymis. These beta-galactosidase labeled neurons may be central autonomic center related to the integration and modulation of reflex control linked to the sensory and motor system monitoring the internal environment. These observations provide evidence for previously unknown projections from epididymis to spinal cord and brain which may be play an important neuroanatomical basic evidence in the regulation of epididymal function.

Ultrastructural Analysis of GABA- and Glycine-Immunoreactive Nerve Terminals on Jaw-Closing and Jaw-Opening Motoneurons in the Rat / 대한해부학회지

Previous studies have shown that inhibitory synaptic inputs are different between in spinal and trigeminal motor systems and activities of jaw closing and opening alpha motoneurons are different during a chewing cycle. This study examined the distribution of inhibitory synapses made on masseter and digastric motoneurons by using retrograde tracing of wheat germ agglutinin conjugated to horseradish peroxides (WGA-HRP) combined with postembedding immunogold labeling on serial ultrathin sections.Many boutons IR (immunoreactive) to GABA and/or glycine were found to appose on two kinds of motoneurons, which were containing pleomorphic vesicles (a mixture of round, oval and flattened vesicles) and exhibited symmetrical synaptic contacts on the somata. Packing density and synaptic covering % were higher in digastric than in masseter motoneurons. Of 703 boutons apposing on 12 masseter motoneurons, 6.08+/-3.51, 29.67+/-8.89 and 17.78+/-5.22% were IR to GABA only, glycine only, and both GABA and glycine, respectively. Of 637 boutons apposing on 11 digastric motoneurons, 6.37+/-4.64, 19.74+/-8.25 and 12.01+/-5.38% were IR to GABA only, glycine only, and both GABA and glycine, respectively. Proportions of glycine IR boutons were higher than that of GABA IR boutons in both masseter and digastric motoneurons. Packing density and proportion of boutons IR to GABA and/or glycine were higher in jaw closing than in jaw opening motoneurons (packing density, 12.03+/-1.58 vs 10.28+/-2.99; proportion of IR boutons, 53.54+/-8.94% vs 38.12+/-9.38% in jaw closing and opening motoneurons, respectively). These results provide ultrastructural evidence that GABA and glycine act as important neurotransmitters for modulation of jaw movement and that proportion of inhibitory synapses is higher in jaw closing than in jaw opening motoneurons.

Localization of sympathetic and sensory nerves innervating heart in the cat using HRP and WGA-HRP as neuronal tracers / 대한해부학회지

The origin of sympathetic and sensory nerves innervating heart in the cat was investigated using HRP (Horseradish peroxidase) and WGA-HRP (Wheat germ agglutinin-horseradish peroxidase) as neuronal tracers. The neural tracers were injected into subepicardial layer and myocardium of the right atrium, left atrium, right ventricle and left ventricle, respectively. Labeled sympathetic neuronal cell bodies were found in superior cervical ganglia, middle cervical ganglia, stellate ganglia and 4th and 5th thoracic ganglia, mainly in middle cervical ganglia and stellate ganglia. Heavier labeled neuronal cell bodies were found in the middle cervical ganglia and stellate ganglia when the neural tracers were injected into left atrium, right ventricle and left ventricle. Labeled sensory neuronal cell bodies were found in nodose ganglia and T1-T6 spinal ganglia, mainly in T1-T5 spinal ganglia. Heavier labeled neuronal cell bodies were found in the nodose ganglia when the neural tracers were injected into left atrium and right ventricle. These results may provide a neuroanatomical data on origin of sensory nerves innervating the heart of the cat.

Quantitative Analysis of GABA and/or Glycine Like Immunoreactive Synaptic Terminals on Masseter gamma-motoneurons for Jaw Closing Muscle of the Rat / 대한해부학회지

The distribution of GABA and/or glycine like immunoreactive nerve terminals on the soma of the masseteric gamma motoneurons were investigated using retrograde tracing of WGA-HRP (wheat germ agglutinin conjugated horseradish peroxidase) and postembedding immunogold labeling methods in serial ultrathin sections. Quantitative analysis of 140 nerve terminals apposing on somata of gamma motoneuron size less than 21 mm in average diameter was performed. The results obtained were as follows. 1. Synaptic covering % of apposing nerve terminals was 21.45+/-11.48% and packing density was 12.85+/-6.17. 2. Nerve terminals immunoreactive (IR) to GABA or glycine were F type containing pleomorphic vesicles with round shape predominant. Majority of nerve terminals immunonegative to GABA or glycine were S type containing spherical vesicles and few of them were F type. 3. 11.42+/-10.00% of examined nerve terminals were IR to GABA only, and 12.71+/-9.85% were IR to GABA and glycine, and 15.21+/-9.58% were IR to glycine only. 4. Synaptic covering % of nerve terminals IR to glycine only was highest (4.58+/-4.50%), followed in order by GABA and glycine (3.18+/-2.77%), and GABA only (2.38+/-2.06%). 5. Among all terminals, immunonegative nerve terminals (60.66+/-14.65%) were much more than nerve terminals immunoreactive to GABA and/or glycine (39.34+/-14.65%) These results show that inhibitory synaptic input and synaptic organization of the masseteric gamma motoneurons reveal characteristic features in contrast to that of alpha motoneurons and which may correlated to the electrophysi-ological characteristics of masseteric gamma motoneurons.

The Localization of Efferent and Afferent Neurons Innervating the Rat Thymus Using the Neural Tracers / 대한해부학회지

The localizations of efferent and afferent neurons were observed following injection of neural tracers, cholera toxin B subunit (CTB) and wheat germ agglutinin-horseradish peroxidase (WGA-HRP) into the rat thymus with ages. Thirty Sprague-Dawley rats were examined at 3 weeks, 5~6 and 20 months of age. After survival times of 48~96 hours following injection of neural tracers, the rats were perfused and their brain, spinal cord, sympathetic ganglia, dorsal root ganglia and vagal ganglia were frozen sectioned (40 mm). These sections were stained by CTB immunohistochemical and HRP histochemical staining methods, and observed with polarized dark and light microscope. The results were as follows: 1. WGA-HRP and CTB labeled parasympathetic neurons were bilaterally seen in the nucleus ambiguus and medullary reticular formation of medulla with all ages. 2. WGA-HRP labeled sympathetic neurons were bilaterally labeled in superior cervical ganglia, middle cervical ganglia, stellate ganglia and T4-8 sympathetic chain ganglia. The number of labeled sympathetic neurons was increased in the thymus at 20 months of age. According to the aging, sympathetic neuronal processes were more developed, and the nerve fibers were coarse and more branched. 3. WGA-HRP labeled sensory neurons were bilaterally observed within the vagal and C1-6 dorsal root ganglia. The number of labeled sensory neurons was decreased in the thymus at 20 months of age.

Localization of Sensory Neurons Innervating the Rat Intestine Using the Cholera Toxin B Subunit(CTB) and Wheat Germ Agglutinin-Horseradish Peroxidase(WGA-HRP) / 영남의대학술지

The local arrangement of sensory nerve cell bodies and nerve fibers in the brain stem, spinal ganglia and nodose ganglia were observed following injection of cholera toxin B subunit(CTB) and wheat germ agglutinin-horseradish peroxidase(WGA-HRP) into the rat intestine. The tracers were injected in the stomach(anterior and posterior portion), duodenum, jejunum, ileum, cecum, ascending colon or descending colon. After survival times of 48-96 hours, the rats were perfused and their brain, spinal and nodose ganglia were frozen sectioned(40microM). These sectiones were stained by CTB immunohistochemical and HRP histochemical staining methods and observed by dark and light microscopy. The results were as follows: 1. WGA-HRP labeled afferent terminal fields in the brain stem were seen in the stomach and cecum, and CTB labeled afferent terminal fields in the brain stem were seen in all parts of the intestine. 2. Afferent terminal fields innervating the intestine were heavily labeled bilaterally gelalinous part of nucleus of tractus solitarius(gelNTS), dorsomedial part of gelNTS, commissural part of NTS(comNTS), medial part of NTS(medNTS), wall of the fourth ventricle, ventral border of area postrema and comNTS in midline dorsal to the central canal. 3. WGA-HRP labeled sensory neurons were observed bilaterally within the spinal ganglia, and labeled sensory neurons innervating the stomach were observed in spinal ganglia T2-L1 and the most numerous in spinal ganglia T8-9. 4. Labeled sensory neurons innervating the duodenum were observed in spinal ganglia T6-L2 and labeled cell number were fewer than the other parts of the intestines. 5. Labeled sensory neurons innervating the jejunum were observed in spinal ganglia T6-L2 and the most numerous area in the spinal ganglia were T12 in left and T13 in right. 6. Labeled sensory neurons innervating the ileum were observed in spinal ganglia T6-L2 and the most numerous area in the spinal ganglia were T11 in left and L1 in right. 7. Labeled sensory neurons innervating the cecum were observed in spinal ganglia T7-L2 and the most numerous area in the spinal ganglia were T11 in left and T11-12 in right. 8. Labeled sensory neurons innervating the ascending colon were observed in spinal ganglia T7-L2 in left, and T9-L4 in right. The most numerous area in the spinal ganglia were T9 in left and T11 in right. 9. Labeled sensory neurons innervating the descending colon were observed in spinal ganglia T9-L2 in left, and T6-L2 in right. The most numerous area in the spinal ganglia were T13 in left and L1 in right. 10. WGA-HRP labeled sensory neurons were observed bilaterally within the nodose ganglia, and the most numerous labeled sensory neurons innervating the abdominal organs were observed in the stomach. 11. The number of labeled sensory neurons within the nodose ganglia innervating small and large intestines were fewer than that of labeled sensory neurons innervating stomach These results indicated that area of sensory neurons innervated all parts of intestines were bilaterally gelatinous part of nucleus tractus solitarius(gelNTS), dorsomedial part of gelNTS, commissural part of NTS(comNTS), medial part of NTS, wall of the fourth ventricle, ventral border of area postrema and com NTS in midline dorsal to the central canal within brain stem, spinal ganglia T2-L4, and nodose ganglia. Labeled sensory neurons innervating the intestines except the stomach were observed in spinal ganglia T6-L4. The most labeled sensory neurons from the small intestine to large intestine came from middle thoracic spinal ganglia to upper lumbar spinal ganglia.

Estudo comparativo de diferentes conjugados de HRP no estudo das vidas neurais das estruturas bucofaciais / Comparative study of free HRP and HRP conjugates in the examination of the connectivity between orofacial structures and the central nervous system

A utilizaçäo de neurotraçadores tem revolucionado, nas últimas três décadas, o conhecimento sobre o padräo de conexöes entre as estruturas periféricas e o sistema nervoso. Entre os neurotraçadores, a peroxidase do rábano silvestre (HRP) tem sido a mais utilizada. Mais recentemente, combinaçöes desse neurotraçador com a aglutinina do germe do trigo (WGA) ou com a subunidade B da toxina colérica (CTB) têm demonstrado um considerável aumento na captaçäo dos conjugados resultantes (HRP-WGA, CTB-HRP), com melhora sensível na capacidade de traçar conexöes neurais. No presente experimento, foram testados ambos os cojugados e o HRP livre no contexto de um amplo estudo sobre o núcleo do nervo hipoglosso do rato, responsável pela inervaçäo motora da língua. Para isto, foram injetados na metade posterior da língua de cada rato 10 µl de HRP (20 por cento) ou 10 µl de CTB-HRP (0,15 por cento de toxina colérica + 12 por cento HRP) ou 10 µl de HRP-WGA (5 por cento de WGA + 30 por cento HRP). Após um período de sobrevivência de 48 horas, os animais foram perfundidos com uma soluçäo de aldeídos e, posteriormente, os encéfalos foram cortados em criostato (40 µm). Os cortes foram submetidos à reaçäo histoquímica do HRP, utilizando-se a tetrametilbenzidina como cromógeno. Os resultados indicam que, tanto desde o ponto de vista qualitativo como até quantitativo, os conjugados säo mais eficientes para evidenciar conexöes neurais que o HRP livre. Os conjugados HRP-WGA e CTB-HRP apresentam qualidades semelhantes, embora este último seja mais eficiente para evidenciar detalhes na arborizaçäo dendrítica e características morfológicas gerais dos motoneurônicos. Essa superioridade do conjugado CTB-HRP pode estar relacionada com a abundância de gangliosídeos do tipo GM1 na membrana pré-sináptica das junçöes neuromusculares. Esses gangliosídeos formam um grupo de receptores específicos para componentes da subunidade B da toxina colérica, o que potencializa a captaçäo da mesma pelos terminais axônicos

The innervation of the thymus gland in the rat: a HRP study

The marker horseradish peroxidase (HRP) was used to retrogradely label the cell bodies of origin of the nerve fibers responsible for thymic innervation. The thymus was exposed in albino rats under general anesthesia and a conjugate of HRP-WGA was injected bilaterally into the gland parenchyma. After a survival time of 3 days, the animals were anesthetized and perfused transcardially. Their brainstems were removed and serially sectioned and together with the whole sympathetic chains were processed for HRP demonstration. Labeled cell bodies were identified in the brainstem at the level of the retrofacial and ambiguous nuclei bilaterally. In the sympathetic chain, labeled neurons were present from the first cervical to the ninth thoracic ganglion, although they were detected in higher numbers in the superior cervical and stelate ganglia.