Ultrastructural detection of neuronally transported choleragenoid by postembedding immunocytochemistry in freeze-substituted Lowicryl HM20™ embedded tissue

Choleragenoid (cholera toxin B-fragment; CTB) is an anterograde, retrograde and transganglionic neuronal tracer. We describe a method for detecting CTB-labeled neuronal cell bodies, neurites and boutons at the ultrastructural level, using postembedding immunogold techniques on freeze-substituted Lowicryl HM20™ embedded nervous tissue. Primary afferents and motoneurons were labeled by injection of CTB in the dorsal ramus of the C2 spinal nerve of the rat. Following fixation with paraformaldehyde (4%) and glutaraldehyde (0.25%), tissue sections from the spinal cord C2 segment were freeze-substituted and embedded in Lowicryl HM20™ and subsequently processed with postembedding immunocytochemistry for CTB and glutamate. Immunogold particles indicating CTB immunoreactivity were found over primary afferents and motoneurons. In primary afferents in the central cervical nucleus (CCN) and motor nuclei, immunogold labeling was seen in boutons over vesicle-containing axoplasm and to a lesser extent over axoplasm devoid of vesicles, but not over mitochondria or axolemma. In motoneurons, immunogold particles were seen over the Golgi apparatus in the soma and over lysosomes in both soma and dendrites. Quantification of glutamate-like immunoreactivity in 20 CTB-labeled and 20 CTB-negative boutons in the neuropil was found similar, indicating that CTB does not interfere with the immunocytochemical detection of neuronal epitopes such as the transmitter substance glutamate.     

Tau immunoreactivity and SDS solubility of two populations of paired helical filaments that differ in morphology

To further understand the processes that lead to the formation of neurofibrillary tangles from paired helical filaments (PHF) in Alzheimer brains, we studied two morphologically distinct fractions of PHF separated on sucrose density gradient. In a fraction with mostly short and non-aggregated PHF, the majority of filaments could be solubilized in SDS. In a fraction containing primarily PHF aggregated into clusters or bundles, sometimes resembling neurofibrillary tangles, filaments were less soluble in SDS. Immunogold labelling with a panel of tau-immunoreactive antibodies demonstrated that N-terminal epitopes of tau were preserved in the short filaments, but were reduced or absent in aggregated filaments. In contrast, C-terminal epitopes were present in both fractions. Furthermore, the accessibility of the microtubule-binding domain to immunolabelling was markedly impaired in short and non-aggregated filaments compared to aggregated filaments. These results are consistent with proteolytic degradation of the N-terminal epitopes and preservation of the C-terminal epitopes and the microtubule-binding domain of tau in the aggregated filaments. Partial proteolysis may be involved in the generation of aggregated PHF in neurofibrillary tangles.     

Immunohistochemical, in situ hybridization, and ultrastructural localization of SARS-associated coronavirus in lung of a fatal case of severe acute respiratory syndrome in Taiwan

This article describes the pathological studies of fatal severe acute respiratory syndrome (SARS) in a 73-year-old man during an outbreak of SARS in Taiwan, 2003. Eight days before onset of symptoms, he visited a municipal hospital that was later identified as the epicenter of a large outbreak of SARS. On admission to National Taiwan University Hospital in Taipei, the patient experienced chest tightness, progressive dyspnea, and low-grade fever. His condition rapidly deteriorated with increasing respiratory difficulty, and he died 7 days after admission. The most prominent histopathologic finding was diffuse alveolar damage of the lung. Immunohistochemical and in situ hybridization assays demonstrated evidence of SARS-associated coronavirus (SARS-CoV) infection in various respiratory epithelial cells, predominantly type II pneumocytes, and in alveolar macrophages in the lung. Electron microscopic examination also revealed coronavirus particles in the pneumocytes, and their identity was confirmed as SARS-CoV by immunogold labeling electron microscopy. This report is the first to describe the cellular localization of SARS-CoV in human lung tissue by using a combination of immunohistochemistry, double-stain immunohistochemistry, in situ hybridization, electron microscopy, and immunogold labeling electron microscopy. These techniques represent valuable laboratory diagnostic modalities and provide insights into the pathogenesis of this emerging infection.     

Ischemic disruption of glutamate homeostasis in brain: quantitative immunocytochemical analyses

More than 10 years ago, it was shown by microdialysis that the excitatory transmitter glutamate accumulates in the interstitial space of brain subjected to ischemic insult. This was one of the key observations leading to the formulation of the `glutamate hypothesis' of ischemic cell death. It is now assumed that even a transient glutamate overflow may set in motion a number of events that ultimately cause cell loss in vulnerable neuronal populations. The aim of the present review is to discuss the intracellular changes that underlie the dysregulation of extracellular glutamate during and after ischemia, with emphasis on data obtained by postembedding, electron microscopic immunogold cytochemistry. While the time resolution of this approach is necessarily limited, it can reveal, quantitatively and at a high level of spatial resolution, how the intracellular pools of glutamate and metabolically related amino acids are perturbed during and after an ischemic insult. Moreover, this can be done in animals whose extracellular amino acid levels are monitored by microdialysis, allowing a direct correlation of extra- and intracellular changes. Immunogold analyses of brains subjected to ischemia have identified dendrites and neuronal somata as likely sources of glutamate efflux, probably mediated by reversal of glutamate uptake. The vesicular glutamate pool has been found to be largely unchanged after 20 min of ischemia. Ischemia causes an increased glutamate content and an increased glutamate/glutamine ratio in glial cells, as revealed by double immunogold labelling. This argues against the idea that glial cells contribute to the extracellular overflow of glutamate in the ischemic brain.     

包埋前免疫电镜双标技术在神经解剖学研究中的应用

目的　在超微结构水平观察两种神经递质在纤维终末内的共存或一种神经递质与其相应受体之间的关系。　方法　包埋前免疫电镜双重标记技术———酶标法和免疫金 银标记法相结合的方法。　结果　在免疫反应双重标记的纹状体切片上 ,电镜下观察到大量的SP样 (过氧化物酶免疫反应产物 )阳性终末和SP受体 (SPR ,免疫金 银标记颗粒 )样阳性神经元的胞体和树突 ,同时可见部分SP样阳性轴突终末分别与SPR样阳性神经元的胞体或树突形成对称性轴 体或轴 树突触联系。而在三叉神经脊束核尾侧亚核切片上 ,电镜下可观察到大量的两种囊泡膜谷氨酸转运体 ,即DNPI样 (过氧化物酶免疫反应产物 )和VGluT1样 (免疫金 银标记颗粒 )阳性轴突终末 ,同时还观察到DNPI样和VGluT1样双标的轴突终末与阴性树突形成非对称性突触。　结论　包埋前免疫电镜双重标记技术敏感性较高 ,组织的抗原性保存好 ,特别是在神经解剖学研究中 ,用于研究两种神经递质在同一个细胞或终末内的共存或分析神经递质与其相应受体之间的联系中有独到之处。     

GABAergic boutons establish synaptic contacts with the soma and dendrites of cuneothalamic relay neurons in the rat cuneate nucleus

This study investigates the synaptic relation between -aminobutyric acid-immunoreactive (GABA-IR) and cuneothalamic relay neurons (CTNs) in the rat cuneate nucleus. Retrograde transport of wheat germ agglutinin conjugated with horseradish peroxidase complex (WGA-HRP) was used to label CTNs while anti-GABA immunogold serum was used for the detection of GABA-IR boutons associated with CTNs. With these procedures, immunogold-labelled GABA-IR boutons were found to form axosomatic, axodendritic and axospinous synapses with the WGA-HRP-labelled but immunonegative CTNs. Quantitative estimation showed that the mean ratios of GABA-IR to GABA-immunonegative boutons making synaptic contacts with somata, proximal dendrites, and distal dendrites were 47.9%, 49.1% and 34.7%, respectively. Statistical analysis showed that the incidence of GABA-IR boutons on the somata and proximal dendrites of CTNs was significantly higher than on the distal dendrites. Our results indicate that GABA is the primary inhibitory neurotransmitter in the cuneate nucleus, thereby emphasizing the importance of postsynaptic inhibition on cuneothalamic relay neurons.     

Synaptic organization of excitatory and inhibitory boutons associated with spinal neurons which project through the dorsal columns of the cat

The cell bodies and proximal dendrites of postsynaptic dorsal column neurons were examined for synaptic boutons which displayed immunoreactivity for the principal excitatory and inhibitory neurotransmitters, glutamate and GABA. The neurons were labelled by retrograde transport of horseradish peroxidase and GABA or glutamate-containing boutons were revealed by performing postembedding immunogold reactions on electron microscope sections. Five neurons were examined and all of them were postsynaptic to boutons which contained either GABA or glutamate. Quantitative analysis of two of the cells revealed that more than 90% of the synaptic profiles associated with them displayed immunogold reactions for these transmitters. Analysis of series of alternate sections, which were reacted for either GABA or glutamate, showed that there was no overlap in the populations of immunoreactive boutons. Furthermore, GABA and glutamate immunoreactions were associated with boutons which had different morphological characteristics. In addition, some large glutamate-enriched boutons were postsynaptic to small boutons which displayed immunogold reactions for GABA. This study demonstrates morphological bases for direct excitation, postsynaptic inhibition and presynaptic inhibition of postsynaptic dorsal column cells.     

Expression of Nerve Growth Factor in Human Pancreatic β Cells

Nerve growth factor (NGF) is an important modulator of rat pancreatic β-cell physiology in vitro. In this study, we analysed the expression of NGF, TrkA and insulin in human pancreatic islets from normal, ductal adenocarcinoma and insulinoma-afflicted samples, using double immunofluorescent labelling and confocal microscopy.

We found that in normal human pancreas, insulin and NGF are co-expressed in β cells. Moreover, similar to previous observations in rat, the high affinity NGF receptor TrkA is also expressed in β cells.

Pancreatic β cells in normal islets from adenocarcinoma and mucinous cystadenocarcinoma patients also expressed NGF. In 2 out of 15 exocrine tumour samples, NGF was detected also in the tissue surrounding the islets, while 2 out of 13 adenocarcinoma tumours expressed this growth factor.

In five insulinoma samples, we observed weaker immunofluorescent labelling of insulin and NGF in the neoplastic tissue, compared to the islets not afflicted by the tumour, which may be a consequence of increased hormone secretion rate.

We demonstrate that human β cells express TrkA and NGF. These findings are consistent with the hypothesis that NGF modulates insulin secretion through a paracrine/autocrine loop, similar to the one observed in cultured rat β cells.     

Localization of Ureaplasma Urealyticum on Human Spermatozoa

Semen specimens were collected from 20 normal fertile men and 20 unexplained infertile men with U reaplasma urealyticum (U.U.) in semen. Spermatozoa of both groups were examined by immunogold technique and immunofluorescence test. A number of gold particles of the U. U. adhered to the sperm surface of infertile men were observed. Strong specific fluorescence was noticed on the sperm surface of infertile men, mostly on the midpiece and/ or postacrosomal region. A significant increase of sperm morphological abnormality, especially the swollen midpiece, the coiled tail, and head-tail angalation sperms was observed in infertile group. The sperm motility in infertile group is dramatically lower than that infertile group, It is hypothesized that teratospermia, poor motility and interference with sperm-ovum interaction might be the possible mechanisms for male infertility caused by Ureaplasma urealyticum.     

Subcellular localization of β-catenin and APC proteins in colorectal preneoplastic and neoplastic lesions

Adenomatous polyposis coli (APC) is a tumor suppressor gene whose main function is the destabilization of β-catenin, a key effector of the Wnt signaling pathway. This gene is defective in familial adenomatous polyposis (FAP), a dominantly inherited disease, but inactivation of APC has been reported also in most sporadic colorectal tumors and it is considered an early event in colorectal tumorigenesis. The aim of the present study was to evaluate the intracellular ultrastructural distribution of β-catenin and APC proteins in epithelial cells of normal colorectal mucosa, aberrant crypt foci (ACF, an early premalignant lesion) and cancer. We used the immunogold electron microscopic method to identify both proteins. Normal colonic epithelial cells showed a strong membranous expression of β-catenin and lacked cytoplasmic and nuclear expression. Normal cells showed APC localization pattern characterized by diffuse nuclear expression and along the plasma membrane. In ACF and in carcinoma an absent or reduced membranous expression of β-catenin was associated with an increased nuclear and cytoplasmatic expression. In aberrant crypt foci and carcinoma, APC was evident inside the nucleus and at the level of cell-cell junctions, but it was decreased in the cytoplasm. This method allowed the accurate localization of proteins of the Wnt signaling pathway in the early steps of colorectal carcinogenesis. The similar pattern of subcellular distribution of APC and β-catenin in dysplastic ACF and colorectal cancer suggests that ACF are precursor lesions of sporadic and FAP-associated colorectal carcinoma.