Experimental studies of mechanisms involved in methods demonstrating axonal and terminal degeneration

Factors influencing the consistency and specificity of the staining of neuronal degeneration products were studied in brain sections by varying systematically the composition of solutions used in the steps which are common to the degeneration methods. The formation of nuclei of metallic silver was determined either by physical development of 110Ag, after dissolving reducible silver by acetic acid. In degenerating axons metallic silver nucleic are formed by their own reducing groups in the first (acid) and in the second (alkaline) impregnating bath. The first impregnation turned out to be sufficient to produce complete staining of degenerating axons. The reducing capacity of normal axons and myelin can be suppressed by oxidation or by lowering the pH of the impregnating solution. Degenerating axon terminals are not able to reduce silver ions in either of the impregnating baths. Rather, the metallic silver nuclei initiating their staining are formed in the Nauta reducer by interaction of its reducing agent (formol) with silver ions which had been trapped in the tissue during the impregnation. Thus the nuclei are enlarged to microscopic visibility by a nonstandardized physical developer coming about from the Nauta reducer and the silver ions transferred with the sections. In this reaction catalytic sites in degenerating terminals as well as ammonium ions and the alkali reserve of the tissue play an important role. On the basis of the present results it was possible to stabilize the conditions for staining degenerating axons and degenerating axons terminals in two separate staining procedures detailed in following papers.     

Experimental Studies of Mechanisms Involved in Methods Demonstrating Axonal and Terminal Degeneration

Factors influencing the consistency and specificity of the staining of neuronal degeneration products were studied in brain sections by varying systematically the composition of solutions used in the steps which are common to the degeneration methods. The formation of nuclei of metallic silver was determined either by physical development or ¹¹⁰Ag, after dissolving reducible silver by acetic acid. In degenerating axons metallic silver nuclei are formed by their own reducing groups in the first (acid) and in the second (alkaline) impregnating bath. The first impregnation turned out to be sufficient to produce complete staining of degenerating axons. The reducing capacity of normal axons and myelin can be suppressed by oxidation or by lowering the pH of the impregnating solution. Degenerating axon terminals are not able to reduce silver ions in either of the impregnating baths. Rather, the metallic silver nuclei initiating their staining are formed in the Nauta reducer by interaction of its reducing agent (formol) with silver ions which had been trapped in the tissue during the impregnation. Thus the nuclei are enlarged to microscopic visibility by a nonstandardized physical developer coming about from the Nauta reducer and the silver ions transferred with the sections. In this reaction catalytic sites in degenerating terminals as well as ammonium ions and the alkali reserve of the tissue play an important role. On the basis of the present results it was possible to stabilize the conditions for staining degenerating axons and degenerating axon terminals in two separate staining procedures detailed in following papers.     

Rapid Bielschowsky silver impregnation method using microwave heating

The Bielschowsky silver impregnation method has been used extensively to demonstrate neuronal processes including dendrites, axons and neurofibrils. In this study, we examined the differences in the time required for and the staining quality of the Bielschowsky method for neuronal processes when microwave heating was used instead of processing at room temperature. For this purpose, a control group of sections stained according to the conventional method at room temperature was compared to an experimental group stained in a microwave oven at 180 W for 2, 4 and 1 min in 2% silver nitrate, ammoniacal silver nitrate and gold chloride, respectively. Light microscopic examination demonstrated that the normal structure was preserved in both groups and that there was no difference in the staining quality between the control and the microwave groups. In addition, staining time for this procedure was reduced to 8 min by using the microwave oven. Our study revealed that microwave irradiation can be used safely for Bielschowsky silver impregnation of neuronal tissues.     

Rapid Bielschowsky silver impregnation method using microwave heating

The Bielschowsky silver impregnation method has been used extensively to demonstrate neuronal processes including dendrites, axons and neurofibrils. In this study, we examined the differences in the time required for and the staining quality of the Bielschowsky method for neuronal processes when microwave heating was used instead of processing at room temperature. For this purpose, a control group of sections stained according to the conventional method at room temperature was compared to an experimental group stained in a microwave oven at 180 W for 2, 4 and 1 min in 2% silver nitrate, ammoniacal silver nitrate and gold chloride, respectively. Light microscopic examination demonstrated that the normal structure was preserved in both groups and that there was no difference in the staining quality between the control and the microwave groups. In addition, staining time for this procedure was reduced to 8 min by using the microwave oven. Our study revealed that microwave irradiation can be used safely for Bielschowsky silver impregnation of neuronal tissues.     

Rapid Bielschowsky silver impregnation method using microwave heating.

The Bielschowsky silver impregnation method has been used extensively to demonstrate neuronal processes including dendrites, axons and neurofibrils. In this study, we examined the differences in the time required for and the staining quality of the Bielschowsky method for neuronal processes when microwave heating was used instead of processing at room temperature. For this purpose, a control group of sections stained according to the conventional method at room temperature was compared to an experimental group stained in a microwave oven at 180 W for 2, 4 and 1 min in 2% silver nitrate, ammoniacal silver nitrate and gold chloride, respectively. Light microscopic examination demonstrated that the normal structure was preserved in both groups and that there was no difference in the staining quality between the control and the microwave groups. In addition, staining time for this procedure was reduced to 8 min by using the microwave oven. Our study revealed that microwave irradiation can be used safely for Bielschowsky silver impregnation of neuronal tissues.     

A reliable method for demonstrating axonal degeneration shortly after axotomy

A method has been elaborated by which degenerating axons can be selectively impregnated with silver. Based on reconsideration of the physicochemical mechanisms of the degeneration methods it takes advantage of physical developers over the chemical ones. The staining procedure is applied to frozen sections of brains fixed with formol. It consists of 6 steps: (1) pretreatment with alkaline hydroxylamine, (2) washing in acetic acid, (3) impregnation in silver nitrate in the presence of ferric ions, (4) washing in citric acid, (5) physical development, and (6) washing in acetic acid. By electron microscopy silver precipitates by this method are almost entirely restricted to the cytoplasm of dense, degenerating axons, sparing mitochondria and myelin sheaths. No special expertise is required to achieve reproducible results. Large numbers of sections treated simultaneously, and large sections, can be stained uniformly. Light microscopic criteria are described which help diagnose the source of possible failures. Low background staining allows dark field illumination and television image analysis to be applied. The method works at survival times of only 3 to 5 days after axotomy. Hence, degenerating axons and axon terminals can be stained in alternating sections from the same brain using this method and another being described separately, which, using different conditions, demonstrates degenerating axon terminals.     

Histological Technic for Cerebellar Climbing and Mossy Fibers

In order to, avoid disadvantages attendant upon the use of fresh frozen sections, or of block impregnation with silver, in staining climbing or mossy fibers of the cerebellum, Rio Hortega's double impregnation method for nerve fibers is useful. This consists of prolonged formalin fixation prior to cutting frozen sections (which thereafter are easier to cut) and preliminary treatment with ammoniacal aqueous and alcoholic washes, mordanting in pyridine silver, and treatment with pyridine-silver-carbonate. Following this, sections are handled individually through one of several reduction methods after which they may be directly mounted or gold toned.     

Periodic-Acid-Foot Stain for Connective Tissue

A marked increase in reticular argyrophilia may be obtained in the Foot ammoniated silver carbonate technic by interposing a strong periodic acid oxidation, 4% aqueous for 2 hours at 25-27°C., prior to silvering. Sections so oxidized before the silver bath show a histological picture of connective tissue that is stronger than that given by the original technic. Stroma of lymphoid tissues (but not other types) is further intensified by brief (5-10 sec.) passage through aqueous 1.5% uranium nitrate after oxidation but before silver impregnation. The specific action of periodic acid (cleavage of the 1,2-glycol linkage to produce aldehyde radicals) strengthens the premise that the carbonyl radical plays an important part in the phenomenon of connective tissue argyrophilia.     

A Reliable Method for Demonstrating Axonal Degeneration Shortly After Axotomy

A method has been elaborated by which degenerating axons can be selectively impregnated with silver. Based on a reconsideration of the physicochemical mechanisms of the degeneration methods it takes advantage of physical developers over the chemical ones. The staining procedure is applied to frozen sections of brains fixed with formol. It consists of 6 steps: (1) pretreatment with alkaline hydroxylamine, (2) washing in acetic acid, (3) impregnation in silver nitrate in the presence of ferric ions, (4) washing in citric acid, (5) physical development, and (6) washing in acetic acid. By electron microscopy silver precipitates by this method are almost entirely restricted to the cytoplasm of dense, degenerating axons, sparing mitochondria and myelin sheaths. No special expertise is required to achieve reproducible results. Large numbers of sections treated simultaneously, and large sections, can be stained uniformly. Light microscopic criteria are described which help diagnose the source of possible failures. Low background staining allows dark field illumination and television image analysis to be applied. The method works at survival times of only 3 to 5 days after axotomy. Hence, degenerating axons and axon terminals can be stained in alternating sections from the same brain using this method and another being described separately, which, using different conditions, demonstrates degenerating axon terminals.     

Demonstration of centrifugal fibers in the optic chiasma and optic nerves in the rat after lesions of the suprachiasmatic nucleus

After an unilateral destruction of the suprachiasmatic nucleus and with the use of several silver impregnation techniques, degenerating centrifugal fibers were found in both optic nerves. Centrifugal fibers to the retina originate from three different regions of the nucleus and their position in the chiasma are different. In large majority the degenerating fibers were located at the periphery of the optic nerve and were more frequent on the contralateral than on the ipsilateral side to the destroyed suprachiasmatic nucleus. The possibility that our experimental procedure demonstrates the existence of fibers originating not only in the suprachiasmatic nucleus but also in other structures whose efferent fibers pass at this level, is discussed.     

Microwaves Applied to Silver Impregnations with Ammoniacal Silver Carbonate

Techniques for impregnation with ammoniacal silver carbonate provide valuable information on all types of tissue; however, the time investment required to impregnate a few sections has limited their application. We have shortened the impregnation times by using microwaves in techniques for reticular fibers, astrocytes, nerve fibers and chromaffin cells. The results were satisfactory with markedly reduced impregnation time and elimination of nonspecific silver deposits.     

Silver Impregnation of Nerve Fibers in Teeth After Decalcification With Ethylenediaminetetraacetic ACID

The difficulties in impregnating bony tissues, which occur after decalcification with acids or electrolysis are avoided by decalcification with ethylenediaminetetraacetic acid at pH 8.2-8.5. The decalcification of adult human teeth which have been cut to a thickness of 2-5 mm takes 1-2 mo. If frozen sections of the decalcified teeth are impregnated 24 hr in 20% AgNo₃, rinsed through 6 changes of 20% neutralized (CaCO₃) formalin, blotted thoroughly with a cloth and placed in an ammoniated silver solution for 15-20 min, reliable impregnation of nerve fibers is obtained. The stock ammoniated silver solution is prepared by adding concentrated NH₄OH to 10-20 ml of 20% AgNO₃ until the precipitate formed by it is dissolved and then adding a few drops of the silver solution until the first permanent opalescence of the mixture is obtained. From this 2 ml are diluted directly before use with 6 ml of distilled water and 4 drops of concentrated NH₄OH added. The diluted stock solution should be used for few (5-10) sections only. The rest of the technic is done in the routine manner.     

Selective Silver Impregnation of Degenerating Axons In The Central Nervous System

Rat and rabbit brains containing surgical lesions of 5-10 days' duration were fixed in 10% formalin (neutralized with calcium carbonate) for 1 week to 6 months. Frozen sections (15-20 n) were rinsed and then soaked 7 minutes in a 1.7% solution of strong ammonia in distilled water. Subsequent treatment was as follows: rinse; 0.05% aqueous potassium permanganate 5-15 minutes; 0.5% aqueous potassium metabisulfite, 2 changes of 2.5 minutes each; wash thoroughly in 3 changes distilled water; 1.5% aqueous silver nitrate, 0.5-1.0 hr.; 1% citric acid, 5-10 sec.; 2 changes distilled water; 1% sodium thiosulfate, 30 see.; 3 changes distilled water. Each section is then processed separately. Ammoniacal silver solution (450 mg. silver nitrate in 10 ml. distilled water; add 5 ml. ethanol; let cool to room temperature; add 1 ml. strong ammonia water and 0.9 ml. of 2.5% aqueous sodium hydroxide), 0.5-1.0 min. with gentle agitation. Reduction of about 1 minute is accomplished in: distilled water, 45 ml.; ethanol, 5 ml.; 10% formalin, 1.5 ml.; 1% citric acid, 1.5 ml. Rinsing; 1% sodium thiosulfate, 10 sec.; thorough washing followed by dehydration through graded alcohol and 3 changes of xylene or toluene complete the staining process. Normal nerve fibers are slightly stained to unstained, degenerating fibers, black. The treatment in potassium permanganate is critical since too little favors overstaining of normal fibers and too much abolishes staining of degenerating fibers.     

A Reliable and Sensitive Method to Localize Terminal Degeneration and Lysosomes in the Central Nervous System

After reconsidering the physico-chemical mechanisms involved in the so-called degeneration methods for the demonstration of axons and nerve terminals, the method of Eager was fundamentally modified in order to stabilize the staining process. This resulted in a simple and reliable method which stains degenerating terminals and lysosomes with a high degree of selectivity and sensitivity. Frozen sections 30 to 50μm thick are prepared from material fixed with formaldehyde by cardiac perfusion. The staining procedure consists of 5 steps: 1) alkaline pretreatment (pH 13), 2) silver impregnation, 3) washing, 4) development at pH 5.0-5.5 monitored by an indicator, and 5) washing in acetic acid. Possible faults can be easily detected by their specific effects on the staining results. Primary submicroscopic silver precipitates are localized selectively in the osmiophilic parts of lysosomes and those degenerating presynaptic elements that are surrounded by glial processes. In degenerating axons, precipitates originating from mitochondria can usually be distinguished from terminal degeneration by their different size, shape, or characteristic arrangement. Nonspecific staining is restricted to glial fibrils, erythrocytes, and single cell nuclei. Dark field illumination can be applied routinely and television image analysis can be used for quantitative evaluation because of low background staining.     

Silver impregnation methods for reticulum fibers and reticulin: A re-investigation of their origins and specifity

Summary Maresch (1905) introduced Bielschowsky's silver impregnation technic for neurofibrils as a stain for reticulum fibers, but emphasized the nonspecifity of such procedures. This lack of specifity has been confirmed repeatedly. Yet, since the 1920's the definition of reticulin and studies of its distribution were based solely on silver impregnation technics. The chemical mechanism and specifity of this group of stains is obscure. Application of Gomori's and Wilder's methods to human tissues showed variations of staining patterns with the fixatives and technics employed. Besides reticulum fibers, various other tissue structures, e.g. I bands of striated muscle, fibers in nervous tissues, and model substances, e.g. polysaccharides, egg white, gliadin, were also stained. Deposition of silver compounds on reticulum fibers was limited to an easily removable substance; the remaining collagen component did not bind silver. These histochemical studies indicate that silver impregnation technics for reticulum fibers have no chemical significance and cannot be considered as histochemical technics for reticulin or type III collagen.     

A Modified Silver Impregnation Technique for the Observation of Thick Sections of Lymph Nodes Perfused with Colloidal Carbon

For many years, a variant of the silver impregnation technique of Bielchowsky has been used to study the lymph node because it clearly outlines the various structures which are usually hard to contrast with standard staining methods. Like other variants of silver impregnation, this method blackens the cell nuclei as well as the reticular fibers; however, it inhibits the impregnation of the nuclear chromatin immediately adjacent to fibers. Hence, this variant selectively darkens the lymphoid cell populations of the nodal structures which contain a loose fiber network.

To study the blood vascular network of the lymph node based on perfusion of colloidal carbon, a staining procedure was needed which would contrast nodal structures on thick sections, while allowing the carbon-filled small blood vessels to be distinguished from the impregnated coarse reticular fibers. In an attempt to adapt this variant of Bielchowsky's technique, 10, 20, 40 and 60 nm thick sections from rat nodes, fixed in a solution of Bouin-Hollande for 72 hr, were silver impregnated with serial dilutions (1:2 to 1:128) of the ammoniacal silver solution. Forty-micrometer thick sections impregnated with a 1:16 dilution of the original silver solution at 37 C and for 30 min provided the best results for the conditions.     

Distribution of reticulospinal fibers and theri endings in the lumbar segments of the rat spinal cord]

After stereotaxis lexions in the nucleus reticularis gigantocellularis of the modulla oblongata and nucleus reticularis pontis caudalis, the distribution of degenerating nerve fibers in the lumbar segments of the spinal cord has been studied by silver impregnation methods of Nauta and Fink-Heimer. Degenerating reticulo-spinal fibers and fragments of axonal terminations were found in the area of n. motorius ventro-medialis and n. motorius ventro-lateralis, as well as partly in n. motorius dorso-lateralis close to motoneurons and their dendrites. Mainly they pass into layers VII and VIII. This fact indicates the existence of direct-reticulo-motoneuronal synaptic connections in rats, which coincides with electrophysiological data.     

Tangential organization of the infragranular fiber plexus in rat cerebral cortex

Cylindrical lesions (diameter 300-500 microns) were formed by poking needles into various parts of the cerebral cortex of adult albino rats. Degenerating axons were visualized in horizontal sections through the 'flattened' cortex using the silver impregnation method of Gallyas et al. [Stain Technol. 55: 291-297 (1980)] which stains degenerating axoplasm. The density and distribution of tangentially oriented axons were evaluated in the infragranular layers by TV image analysis. The sampling fields were concentrically arranged around the lesion at distances of 200, 400, 700 and 1,100 microns. The results indicate that the distribution patterns of degenerating (associational) axons covary with the cytoarchitectonic regions into which the lesions were placed. In the motor cortex, the majority of axons run in the antero-posterior direction. The density is generally lower around lesions in frontal regions than in parietal regions. The most extended degeneration was found around lesions near the border of or within the retrosplenial cortex, indicating an exceptionally strong internal connectivity in this area. Since only few degenerating axons were seen around lesions in the center of area 17, the high density of myelinated axons in the primary visual cortex seems to be due to fibers that originate in peristrate areas. It is concluded that the number and extension of fibers that degenerate tends to covary with some aspects of cortical architecture, but it is not area-specific.     

Differential Impregnation of Degenerating Nerve Fibers in Paraffin-Embedded Material

The silver method of Nauta and Gygax (1951) has been used on paraffin embedded material to give a result closely comparable to that obtained by Nauta and Gygax (1954) on frozen sections. It has been found that pyridine plays an important part in suppressing the impregnation of normal fibers in paraffin embedded material and that this sup pression can be augmented by the use of some of the higher methylated derivatives of pyridine, particularly 2,4,6-trimethyl pyridine (collidine).     

SILVER IMPREGNATION OF ULTRATHIN SECTIONS FOR ELECTRON MICROSCOPY

A new procedure is described for silver impregnation of thin sections for electron microscopy. Sections of various tissues, fixed in OsO₄ and embedded in methacrylate, were treated with an ammoniacal silver solution, directly or after oxidation with periodic acid or hydrogen peroxide. After OsO₄ fixation all cellular membranous systems exhibit a primary argentaffinity probably due to the reduction of ammoniacal silver solution by the reduced osmium bound to unsaturated lipids. Bleaching the sections with hydrogen peroxide removes the argentaffinity of protoplasmic structures. Treatment of the sections with periodic acid results in decreased argentaffinity of protoplasmic components while the argentaffinity of metaplasmic structures is greatly enhanced. The latter procedure appears particularly useful for enhancing the contrast of basement membranes.