Improved Gold Chloride Procedure for Nerve Staining in Whole Mounts of Rat Corneas

The purpose of this study was to modify the gold chloride procedure for studies of total innervation in corneal whole mounts to provide a decrease in nonspecific background staining and to eliminate the progressively deteriorating stain quality of standard gold chloride techniques. Modifications included use of cryo-protective agents, mechanical removal of Descemet's membrane-endothelium complex prior to fixation, treatment with alpha amylase, and halting the reduction of gold chloride to metallic gold using Kodak rapid fixer with hardener. Rat corneas were stored at-70 C in O. C.T. compound. The Descemet's membrane-endothelium complex was removed after thawing, and corneas were fixed in 4% NaPO₄-buffered paraformaldehyde with 8% sucrose. Fixed corneas were incubated in NaPO₄-buffered saline containing alpha amylase, placed in 100% lemon juice, then in 1% gold chloride solution, transferred to glacial acidic acid, placed in rapid fixer, rinsed in NaPO₄-buffered saline, and dehydrated in graded alcohols. Flat mounts of whole corneas were examined using contralateral corneas as controls. Freezing corneas in O. C.T. compound, removal of the Descemet's membrane-endothelium complex, and treatment with alpha amylase reduced nonspecific background staining compared to controls. Treatment with Kodak rapid fixer prevented the deterioration of staining quality for at least 8 months. These improvements allow the gold chloride technique to be used with immunohisto-chemical procedures where the reaction products would be obscured by background staining.     

Suppression of Costaining of Nonnervous Tissue in Protargol Technics

If, in the procedure of staining nerve fibers in mounted paraffin sections with Protargol according to Bodian, the reduction after toning with gold chloride is executed in a solution of 3-6 drops of aniline oil in 100 ml of 50% alcohol instead of in the prescribed oxalic acid solution, the selectivity of the staining of peripheral nerves is increased. This is effected by a reduction in the intensity of the staining of nonnervous tissue elements. However, at the same time the staining of nonnervous tissue is richer in details and consequently more satisfactory from a histological point of view than it is according to the original method of Bodian or the modification of this method by Ziesmer (1951).     

Characterization of Merkel cells and mechanosensory axons of the rat by styryl pyridinium dyes

Summary The epidermal Merkel cells and their sensory innervation serve tactile sensation in vertebrates. In this study the fluorescent cationic mitochondrial dye, 4-(4-diethylaminostyryl)-N-methylpyridinium iodide (4-Di-2-ASP), which has recently been used as a vital stain for motor and autonomic nerve terminals, was tested for its ability to stain Merkel cells and sensory fibers in the snout of the rat. Brightly-fluorescent structures resembling Merkel cells as well as nerve fibers and their terminations were evident in whole mounts of the vibrissal follicle. Unilateral denervation of the vibrissal follicles soon after birth resulted in a staining pattern remarkably similar to that obtained after labelling of the Merkel cells selectively with the fluorescent marker quinacrine, but all fiber staining was abolished. Likewise, in the separated epidermis of other skin regions, including the hairy and glabrous skin of the nose, the staining pattern revealed by 4-Di-2-ASP was indistinguishable from that obtained by quinacrine fluorescence. These results indicate that certain styryl pyridinium dyes may be used as vital stains for epidermal Merkel cells as well as cutaneous mechanosensory axons.     

The Use of Gold Chloride to Stain Developing and Adult Neuromuscular Junctions in the Insect

Individual insect muscle fibers, whose neuromuscular junctions have been stained with a modification of Ranvier's gold chloride method, can be dissected free and mounted whole if the muscle is prefixed in aldehydes. The neuromuscular junctions along the length of the individual fibers are well delineated and can be measured and counted. Effective procedures include fixation with glutaraldehyde buffered to low pH with sodium citrate, or glutaraldehyde and paraformaldehyde combined in phosphate buffer at neutral pH, followed by exposure to citric acid and to gold chloride. The method is convenient, and could be useful for the study of arthropod neuromuscular junctions in general, since their nerve terminals do not release acetylcholine as a transmitter and cannot be stained by the more commonly used cholinesterase methods.     

The use of gold chloride to stain developing and adult neuromuscular junctions in the insect

Individual insect muscle fibers, whose neuromuscular junctions have been stained with a modification of Ranvier's gold chloride method, can be dissected free and mounted whole if the muscle is prefixed in aldehydes. The neuromuscular junctions along the length of the individual fibers are well delineated and can be measured and counted. Effective procedures include fixation with glutaraldehyde buffered to low pH with sodium citrate, or glutaraldehyde and paraformaldehyde combined in phosphate buffer at neutral pH, followed by exposure to citric acid and to gold chloride. The method is convenient, and could be useful for the study of arthropod neuromuscular junctions in general, since their nerve terminals do not release acetylcholine as a transmitter and cannot be stained by the more commonly used cholinesterase methods.     

Improved gold chloride staining method for anatomical analysis of sensory nerve endings in the shoulder capsule and labrum as examples of loose and dense fibrous tissues

Consistency in gold chloride staining is essential for anatomical analysis of sensory nerve endings. The gold chloride stain for this purpose has been modified by many investigators, but often yields inconsistent staining, which makes it difficult to differentiate structures and to determine nerve ending distribution in large tissue samples. We introduce additional steps and major changes to the modified Gairns’ protocol. We controlled the temperature and mixing rate during tissue staining to achieve consistent staining and complete solution penetration. We subjected samples to sucrose dehydration to improve cutting efficiency. We then exposed samples to a solution containing lemon juice, formic acid and paraformaldehyde to produce optimal tissue transparency with minimal tissue deformity. We extended the time for gold chloride impregnation 1.5 fold. Gold chloride was reduced in the labrum using 25% formic acid in water for 18 h and in the capsule using 25% formic acid in citrate phosphate buffer for 2 h. Citrate binds gold nanoparticles, which minimizes aggregation in the tissue. We stored samples in fresh ultrapure water at 4° C to slow reduction and to maintain color contrast in the tissue. Tissue samples were embedded in Tissue Tek and sectioned at 80 and 100 μm instead of using glycerin and teasing the tissue apart as in Gairns’ modified gold chloride method. We attached sections directly to gelatin subbed slides after sectioning with a cryostat. The slides then were processed and coverslipped with Permount. Staining consistency was demonstrated throughout the tissue sections and neural structures were clearly identifiable.     

Simultaneous demonstration of adrenergic and non-adrenergic nerve fibres

Summary A technique for simultaneous demonstration of adrenergic and non-adrenergic nerve fibres is described, using methylene blue staining and fluorescence microscopy after formaldehyde treatment. The procedure is applicable to whole mounts as well as to microtome sections.     

Enkephalin-like immunofluorescence in nerves of the rat iris following systemic capsaicin injection

A network of nerve fibers with an enkephalin-like immunoreactivity was demonstrated in rat iris whole mounts. Systemic administration of capsaicin in doses which caused partial (5 mg/kg) or complete (50 mg/kg) disappearance of substance P-containing fibers in the iris did not cause degeneration of enkephalin-positive nerve fibers. The enkephalin-immunoreactive network seemed intact also after a capsaicin dose of 250 mg/kg. In fact, the fluorescence intensity of the nerve fibers showing enkephalin-immunoreactivity was often increased three days after a capsaicin injection in a dose of 50 mg/kg. The mechanism behind this effect of capsaicin remains to be elucidated, but could be due either to a direct effect on the enkephalin-positive nerves or involve the disappearance of substance P nerves and/or a simultaneous inflammatory response. However, an increased fluorescence intensity of the enkephalin-immunoreactive fibers was sometimes seen also without capsaicin treatment.     

Multisegmental cobalt filling of the dorsal giant fibers in the nervous system of the earthworm,Lumbricus terrestris

Summary The anatomical organization of the two dorsal giant fiber systems of the earthworm Lumbricus terrestris is demonstrated in whole mounts and serial-section reconstructions based on backfillings of the ventral nerve cord with cobalt chloride. Both the medial and lateral fiber systems can be labeled selectively over more than ten body segments. They show a characteristic segmental pattern of collaterals with some modification in tail segments and of dorsal plasma protrusions in the unpaired medial giant fiber presumably representing openings in the myelin sheath. We found no multisegmental cobalt transport in other large neurons of the nerve cord. Cobalt passes through the segmentai septa between consecutive axonal elements of the metameric giant fibers and presumably also through commissural contacts between specific collaterals of the lateral giant fibers. Since these sites of contact are known to represent electrical synapses, cobalt coupling may, in L. terrestris, correlate with functional electrotonic coupling.Abbreviations CL collateral of lateral giant fiber - CM collateral of medial giant fiber - GIN giant interneuron - LGF lateral giant fiber - MGF medial giant fiber - SN segmental nerve     

Lectin and Neuropeptide Labeling of Separate Populations of Dorsal Root Ganglion Neurons and Associated “Nociceptor” Thin Axons in Rat Testis and Cornea Whole-Mount Preparations

As part of a program to explore patterns of innervation by nociceptor-related thin sensory axons in a variety of peripheral regions, we have labeled calcitonin gene-related peptide immunoreactive (CGRP-IR) nerve fibers in whole mounts of rat testicular tunica vasculosa and cornea. Efforts were undertaken to visualize the numerically significant fluoride-resistant acid phosphatase (FRAP)-containing axon population, whose peripheral endings have heretofore remained undemonstrable due to technical limitations of currently available acid phosphatase methods. Various histochemical markers that colocalize with FRAP in dorsal root ganglion (DRG) and spinal cord were examined, and a plant lectin, Griffonia simplicifolia I-B₄, has been identified that not only selectively labels FRAP(+) sensory ganglion cells and central terminals in spinal cord, but also differentially stains a large number of thin axons in testicular and corneal whole mounts. Slender lectin-labeled fibers are abundant in cornea, and are distributed throughout tunica vasculosa preparations unrelated to blood vessels. CGRP-IR axons, in contrast, maintain close adherence to vascular patterns and are more coarse and varicose in appearance.

Lectin staining therefore provides the first practical and specific method for visualization of peripheral FRAP(+) axons consisting principally of sensory C fibers but possibly including a small number of unmyelinated autonomic axons. It should now be feasible, using individual whole-mount preparations from various peripheral nociceptor-innervated tissues, to examine the distributions of both peptidergic and FRAP(+) fibers, which together comprise the vast majority of thin sensory axons. It may then be possible to correlate the observed anatomical patterns with knowledge regarding properties of corresponding physiologically characterized receptive fields.     

Distribution of cholinergic and catecholaminergic nerves in the colon of the rat with aganglionosis.

We compared localization and distribution of putative cholinergic fibers by acetylcholinesterase and of adrenergic fibers visualized by the glyoxylic acid technique in the aganglionic segment using whole mount preparations of aganglionosis rat (AGR) and compared them with those of normal littermates. We also attempted simultaneous staining of acetylcholinesterase (AChE) and catecholamine fluorescence (C-F) on the same whole mount preparations to compare the differences in distribution pattern. All AGR used in this study had narrowed segments of the bowel extending from the distal ileum to the anus, and had no ganglion cells in these narrowed segments. In the intermuscular space, normally occupied with myenteric ganglion, of the narrowed distal colon and rectum, various sizes of nerve bundles and fibers reactive for AChE and C-F appeared to make coarse and irregular networks. These thick nerve bundles appeared to ascend to the proximal colon and disappeared in the cecum. In the distal ileum, almost totally absence of AChE positive nerve fibers, but a few fine C-F fibers, probably associated with blood vessels, were observed. By the method of simultaneous staining of AChE and C-F method in the whole mount preparations, the thick nerve bundles in the narrowed segments showed both of AChE positive and C-F positive. However, there were differences in peripheral fine nerve fibers in the segment; especially numerous perivascular C-F positive nerve fibers, but a few AChE positive ones were found. In the upper aganglionic narrowed segments, greatly diminished numbers of AChE positive and C-F positive nerve fibers were found in the circular muscle layer and in the submucosal layer. In the lower aganglionic narrowed segments, there were thick nerve bundles, forming irregular interlaced network. The role of these extrinsic nerve fibers in aganglionic segments is unclear.     

Possible Involvement of Polysialogangliosides in Nerve Sprouting and Cell Contact Formation: An Ultracytochemical In Vitro Study

In Cichlid fish (Oreochromis mossambicus) primary cell cultures from whole brain and optic tectum, the differentiation-dependent distribution of polysialogangliosides on the outer cell surface has been followed on an ultrastructural level. For this, a two-step labeling technique with the monoclonal mouse antibody Q211, recognizing a polysialoganglioside-associated epitope, followed by a secondary IgM antibody, coupled to colloidal gold sols as an electron-dense marker, has been used. The gold grains are not uniformly distributed over the whole cell surface, but rather are clearly arranged clusters. In cells from freshly hatched larvae, both cell bodies and nerve fibers strongly exhibit the polysialoganglioside epitope on their surface. With progressing development, neuronal cell labeling is more and more restricted to nerve fibers and especially to cellular adhesion zones, including synaptic terminals, thus suggesting a functional involvement of polysialogangliosides in nerve sprouting and initiation of both cell-to-extracellular matrix and cell-to-cell contacts.     

A rapid, sensitive histochemical stain for myelin in frozen brain sections.

A staining technique is described whereby frozen brain sections are incubated in a vehicle containing gold chloride. After 2-4 hr in this solution, both large myelinated bundles and fine individually myelinated fibers are darkly stained. Advantages of this technique over conventional myelin stains include speed, sensitivity, metachromatic staining, and compatibility with formalin-fixed and frozen cut sections. Possible histochemical mechanisms are discussed.     

Enkephalin immunoreactivity in iris nerves: Distribution in normal and grafted irides,persistence and enhanced fluorescence after denervations

Summary The morphology and distribution of nerve fibers showing enkephalin-like immunoreactivity was studied in rat and mouse iris whole mounts. In adult rat, a relatively dense network of varicose fibers was seen throughout the iris. Individual, long, usually smooth fibers were observed running together with non-fluorescent fibers in bundles. Positive nerve fibers were also seen in the ciliary body and the choroid membrane. The fluorescence intensity was normally low. No enkephalin-positive fibers were detected in adult mouse iris.Extirpation or lesioning either one or all the three ganglia known to supply the rat iris with nerve fibers, the superior cervical, the ciliary and the trigeminal ganglia, caused no detectable decrease in amount of enkephalin-positive fibers. However, in irides grafted to the anterior eye chamber of adult recipients, no enkephalin-positive fibers could be observed 2–12 days postoperatively, strongly suggesting that degeneration of these fibers had occurred. When iris grafts were left longer in the eye, nerve fibers with enkephalin-like immunoreactivity reappeared. An increased fluorescence intensity was observed both in the ipsilateral and contralateral iris following extirpation or lesioning all three ganglia and in the ipsilateral iris after extirpation of the ciliary ganglion. Three days after a systemic injection of capsaicin, which causes a permanent disappearance of substance P fibers, the same phenomenon was often observed. This raises the possibility of an interaction between the enkephalin-positive and the substance P fiber systems in the iris.The present experiments thus demonstrate a rich network of enkephalin immunoreactive nerve fibers in the rat iris originating outside the iris but apparently not in the ciliary, trigeminal or superior cervical ganglion.     

Enkephalin immunoreactivity in iris nerves: distribution in normal and grafted irides, persistence and enhanced fluorescence after denervations

The morphology and distribution of nerve fibers showing enkephalin-like immunoreactivity was studied in rat and mouse iris whole mounts. In adult rat, a relatively dense network of varicose fibers was seen throughout the iris. Individual, long, usually smooth fibers were observed running together with non-fluorescent fibers in bundles. Positive nerve fibers were also seen in the ciliary body and the choroid membrane. The fluorescence intensity was normally low. No enkephalin-positive fibers were detected in adult mouse iris. Extirpation or lesioning either one or all the three ganglia known to supply the rat iris with nerve fibers, the superior cervical, the ciliary and the trigeminal ganglia, caused no detectable decrease in amount of enkephalin-positive fibers. However, in irides grafted to the anterior eye chamber of adult recipients, no enkephalin-positive fibers could be observed 2-12 days postoperatively, strongly suggesting that degeneration of these fibers had occurred. When iris grafts were left longer in the eye, nerve fibers with enkephalin-like immunoreactivity reappeared. An increased fluorescence intensity was observed both in the ipsilateral and contralateral iris following extirpation or lesioning all three ganglia and in the ipsilateral iris after extirpation of the ciliary ganglion. Three days after a systemic injection of capsaicin, which causes a permanent disappearance of substance P fibers, the same phenomenon was often observed. This raises the possibility of an interaction between the enkephalin-positive and the substance P fiber systems in the iris.(ABSTRACT TRUNCATED AT 250 WORDS)     

Histochemical profiles of cat intrafusal muscle fibers and their motor innervation

Muscle spindles were examined histochemically in serial transverse sections of cat tenuissimus muscles. The myofibrillar adenosine triphosphatase (ATPase) staining reaction was used to identify nuclear bag1, bag2 and nuclear chain intrafusal muscle fibers. Regional differences in ATPase staining occurred along the bag1 and bag2 fibers but not along the chain fibers. All intrafusal fiber types displayed regional variability in staining for nicotinamide adenine dinucleotide tetrazolium reductase (NADH-TR). Motor nerve terminals were demonstrated along the poles of bag1, bag2 and chain fibers by staining for cholinesterase (ChE). There was no consistent spatial correlation between the intensity of regional ATPase staining along the bag fibers and location, number or type of motor endings. However, most ChE deposits occurred in intrafusal fiber regions that displayed the greatest NADH-TR variability. Some fiber poles or whole intrafusal fibers were devoid of any ChE deposits but their ATPase and NADH-TR content was comparable to that of fibers bearing ChE deposits. The observations suggested that motor nerve fibers per se may not play a major role in determining the histoenzymatic content of intrafusal fibers.     

High resolution neurochemical gold staining method for myelin in peripheral and central nervous system at the light- and electron-microscopic level

Myelin is a multilamellar membrane structure primarily composed of lipids and myelin proteins essential for proper neuronal function. Since myelin is a target structure involved in many pathophysiological conditions such as metabolic, viral, and autoimmune diseases and genetic myelin disorders, a reliable myelin detection technique is required that is equally suitable for light- and electron-microscopic analysis. Here, we report that single myelinated fibers are specifically stained by the gold phosphate complex, Black gold, which stains myelin in the brain, spinal cord, and peripheral nerve fibers in a reliable manner. Electron-microscopic and morphometric analyses have revealed that gold particles are equally distributed in the inner, compact, and outer myelin layers. In contrast to Luxol fast blue, the gold dye stains proteinase-sensitive myelin structures, indicating its selective labeling of myelin-specific proteins. Aiming at defining the target of gold staining, we performed staining in several mouse myelin mutants. Gold complex distribution and myelin staining in MBP^−/−/shiverer mouse mutants was comparable with that seen in wild-type mice but revealed a more clustered Black gold distribution. This gold staining method thus provides a sensitive and specific high-resolution marker for both central and peripheral myelin sheaths; it also allows the quantitative analysis of myelinated fibers at the light- and electron-microscopic level suitable for investigations of myelin and axonal disorders. This study was supported by grants from the International Human Frontier Science Program Organization (HFSPO, to N.E.S.) and the Danone Institute (to N.E.S. and I.Y.E.).     

大鼠脊髓半横断伤联合架桥模型制作及评估

目的:建立大鼠脊髓半横断伤联合架桥模型,为研究脊髓损伤提供动物模型。方法:制作大鼠脊髓半横断伤模型,然后取大鼠前肢正中神经,并于半横断伤两端行正中神经架桥术。术后4周,左心室灌注固定取材,免疫组化染色检测GFAP、RECA、NF-200;另一部分动物行单宁酸-氯化铁灌注;观察移植物内有无血管、血管内有无血流、血管与周边神经纤维的关系。结果:外周神经架桥后4周,移植正中神经贴合于脊髓背侧1/2。移植神经内有RECA阳性的血管存在,而且有血流可以到达移植物内部,且神经纤维(NF-200阳性)与星形胶质细胞(GFAP阳性)关系紧密。结论:大鼠脊髓半横断伤联合正中神经架桥术后,由宿主可以向移植物内长入新生血管,血管有利于神经纤维的存活及生长。本模型为较好的外周神经移植的存活模型,可为进一步的深入研究提供一定的依据。     

Gold enhancement of gold-labeled probes: gold-intensified staining technique (GIST)

In this report we present a staining method in which gold chloride is used to enhance the size of gold colloids. We show the utility of this technique when used in conjunction with small gold colloids, i.e., 5 nm, 4 nm, and 2.6 nm. Post-embedding staining of epoxy-embedded, gold-labeled mouse LM fibroblasts showed that staining with 0.1% gold chloride facilitated the visualization of the smallest gold colloids.