Peggy E. Yates

The demonstration of cholinesterases by the formation of osmium blacks at the sites of Hatchett's brown

SummarySmall amounts of Hatchetts brown (cupric ferrocyanide, Cu2Fe(CN)6·7H2O), deposited at the sites of cholinesterase activity in tissues by the procedure of Karnovsky and Roots (1964), may be enhanced by bridging to osmium through thiocarbohydrazide (TCH). Alternatively, amplification of the deposits may be attained by utilizing the cupric ferrocyanide as a catalyst to effect the oxidative coupling of 3,3′-diaminobenzidine (DAB). The resulting intensely colored osmiophilic polymer is more visible and after osmication, is electron opaque. With these procedures, the light microscope preparations are more permanent than with the Gomori modification of the Koelle and Friedenwald procedure.One of the major advantages of this method is that thick serial plastic sections (1–2 μm) may be taken and readily studied by light microscopy until the required enzymatically stained areas appear. Thus the selection of areas for ultrathin sectioning is facilitated; enzymatically stained structures usually difficult to locate, such as intraepithelial nerve endings, are readily found.The intensification procedures permit shorter incubation times at lower temperatures. This results in diminished tendency of the original amorphous, gel-like Hatchetts brown deposits to coalesce into relatively large cubic crystals.Immediate rinsing of the tissues after the formation of Hatchetts brown with a 0.1 M, pH 7.2, tris (hydroxymethyl)aminomethane (TRIS) buffer prior to intensification with either TCH or DAB was found effective in eliminating some of the smaller background deposits after the longer incubations.Studies in developing mice with an hereditary sensory neuropathy showed severe depletion of acetylcholinesterase activity concomitant with the loss of sensory endings from the rugae of hard palate, confirming the peripheral nature of the neuropathy.High levels of acetylcholinesterase found associated with afferent trigeminal components, especially sensory endings containing clear vesicles, suggest that they may be cholinoceptive.

New methods for the demonstration of lysosomal hydrolases by the formation of osmium blacks

SummaryMethods are described for the direct cytochemical demonstration of the enzymes nonspecific esterase and acid phosphatase based on synthetic substrates which initially deposit Hatchetts brown (cupric ferrocyanide, Cu2Fe(CN)6·7 H2O) at their subcellular sites. The small amounts of Hatchetts brown deposited as a result of the enzymes activity may be intensified by bridging to osmium through thiocarbohydrazide. Alternatively, even greater amplification of the sites of activity may be attained by utilizing the Hatchetts brown as a catalyst to effect the oxidative coupling of 3,3′-diaminobenzidine resulting in the formation of an osmiophilic indamine-type polymer.One of the major advantages of this new approach is that it permits the study of acid hydrolase localization without lead in the incubation medium. Studies were performed with these methods having identical incubation media except for synthetic substrate in many different cell types and tissues. They verify a frequent nonlysosomal localization for acid phosphatase and the heterogeneity of lysosomes and lysosomal populations with respect to hydrolase content.These methods give information obtained by direct cytochemical observation an advantage not previously held, in comparison with information from cell-fractionation cytochemical or biochemical studies. Initial studies with these methods on many tissues reinforce previous suggestions of the involvement of acid hydrolases in extralysosomal sites in subcellulur anabolic processes.

Peripheral neuropathy in mouse hereditary diabetes mellitus. I. Comparison of neurologic, histologic, and morphometric parameters with dystonic mice.

SummaryC57BL/KsJdb/db inbred mice have an hereditary autosomal recessive disease resembling in some respects maturity onset human diabetes mellitus. At 8–11 months of age, they displayed intermittent symptoms suggestive of a mild sensory neuropathy. These symptoms consisted of adduction of their hind limbs and flexing hind paws when raised by the tail, and inability to maintain their position on the roto wheel. Peripheral nerves and sensory ganglia of the diabetic mice were compared with those of the unafflicted littermates and studied with respect to Schwann cell counts and myclinated nerve fiber diameter measurements. In addition, teased fibers of peripheral nerves were compared for obvious changes in internodal distance and demyelination. Chromatolytic neurons were more abundant in lumbosacral spinal ganglia of diabetic mice than in corresponding ganglia of controls or in more anterior spinal ganglia and trigeminal ganglia of diabetics. Histologic studies showed an increase in Schwann cell counts in longitudinal sections of peripheral nerves. A similar but larger increase was observed in peripheral nerves of mice affected with an hereditary sensory neuropathy, dystonia musculorum. A small but general decrease in myelinated fiber diameter was observed in sensory and motor nerves.

The demonstration of arylsulfatases with 4-nitro-1,2-benzenediol mono(hydrogen sulfate) by the formation of osmium Blacks at the sites of copper capture

SummaryA new method is described for the direct cytochemical demonstration of lysosomal arylsulfatases utilizing a synthetic substrate, 4-nitro-1,2-benzenediol mono)hydrogen sulfate), and a copper capture reaction. A small amount of Hatchetts brown (cupric ferrocyanide, Cu2Fe(CN)6·7 H2O) formed at the subcellular sites of copper capture is then utilized as a heterogeneous catalyst to effect the oxidative polymerization of 3,3′-diaminobenzidine which results in the formation of an insoluble, highly colored osmiophilic indamine polymer at the sites of enzymatic activity. The reaction product even at this stage prior to osmication is highly visible. It is readily seen with a light microscope in 50 μm sections of fixed tissues prepared with a mechanical chopper or in 10 micron cryostat sections treated for arylsulfatase activity. Upon osmication, an electron-opaque osmium black is formed which is much less soluble than the products of either the lead or barium capture reactions currently used for the demonstration of arylsulfatase with the electron microscope. The selection of areas of plastic-embedded tissues for ultrathin sectioning is facilitated by the ready visibility of these osmium black end products on 1–2 μm plastic sections which can be studied with the light microscope.This method gives permanent specimens demonstrating arylsulfatases A or B in lysosomes and autophagic vacuoles. In addition, enzyme activity is seen occasionally in the Golgi region or lamellae of certain cells believed to be elaborating sulfated products. In these instances, it may be demonstrating sulfotransferase activity.

Cytochemical correlates of structural sexual dimorphism in glandular tissues of the mouse

SummaryCirculating androgens are known to effect a sexual dimorphism of the submandibular gland and kidney of the mouse. Enzyme histocytochemical differences that correlate with these structural changes have been the subject of much study, especially in the kidney. In the present study, emphasis was placed on the hypogonadic effects of diabetes mellitus on the submandibular gland and kidney of C57BL/KsJ db/db inbred mice with an autosomal recessive disease resembling maturity onset human diabetes mellitus. These glands of adult diabetic mice of both sexes were compared with those of unafflicted heterozygous littermates. The mitochondrial cytochrome oxidase and peroxisomal and cytoplasmic catalase were studied in their submandibular glands and kidneys. The parasympathetic innervation of the submandibular glands was studied by a histochemical method for acetylcholinesterase. The extensive differentiation of striated ducts of the submandibular gland into granular tubules in the postpubertal male mouse was readily evident with the cytochrome oxidase procedure. This differentiation resulted in ductal staining patterns characteristic of the sexes. Alteration of these patterns suggested that demasculinization or feminization was occuring in the male diabetic mice and that masculinization or virilization (defeminization) was occurring in the female diabetics. Similarly, in kidney, study of the parietal epithelium of Bowmans capsule revealed feminization in the male diabetics and masculinization in the female diabetics. With the catalase procedure, a dramatic sexual dimorphism was observed in the kidneys of the heterozygous unafflicted mice. Peroxisomal staining of epithelial cells of the proximal convoluted tubules was much more intense in the outer medulla of the male than of the female. In kidneys of the diabetics, the staining patterns again suggested that feminization of the male and masculinization of the female kidneys had occurred. On the other hand, neither a sexual dichotomy nor effects due to diabetes could be observed in the characteristic catalase staining observed in the luminal epithelial cells of submandibular gland distal ducts. The parasympathetic innervation of the submandibular gland, as revealed by the acetylcholinesterase method, was also markedly sexually dimorphic in the unafflicted mice. This was due to the more extensive innervation of the larger granular ducts characteristic of male than of the smaller striated ducts of the female. As a result of diabetes, the innervation and duct size decreased in the submandibular gland of the male, suggesting feminization, whereas they increased in the female suggesting masculinization. These changes were consistent with those observed in submandibular with the cytochrome oxidase procedure. Attempts were made to interrelate all of the enzyme histochemical changes observed in submandibular gland and kidney with the weights of these glands, sex, gonadal weights, diabetic status and urinary protein excretion. Generally, significant differences were recorded which suggested that the feminization of the submandibular gland and kidney in the diabetic male mice, and their masculinization in the female diabetics, were due to the hypogonadism of the disease.

The formaldehyde-sensitivity of acid phosphatases involved in osteogenesis and odontogenesis in the rat

Abstract The activity of acid phosphatases in odontoblasts, secretory ameloblasts and osteoblasts is selectively inhibited by pretreating 10 μm freeze-dried whole-body sections of 10-day-old rats with formaldehyde. The acid phosphatase activity of relatively large trigeminal ganglion cells is also formalin-sensitive. The localization of formalin-sensitive acid phosphatases in cells of the above types which have relatively high RNA content and a paucity of lysosomes, is supportive of an extra-lysosomal localization previously suggested for these enzymes.

Demonstration of the Bacterial-Biomaterial Interface in Implant Specimens

Bacterial infection can be a problem associated with biomaterial implants especially with the total artificial heart or other cardiovascular prostheses such as ventricular assist devices, intravenous catheters, ventriculo-atrial shunts, pacemaker electrodes and, rarely, prosthetic heart valves. Bacterial commensals such as Staphylococcus epidermidis, which is ordinarily non-infective in human skin and mucous membranes, is now recognized as an opportunistic pathogen of biomaterial implants, particularly cardiovascular prostheses. In these implantations the S. epidermidis undergoes transformation to produce mucoid or polysaccharide extracellular coating substances. The latter promote bacterial adherence to biomaterial surfaces and protect the bacteria to some extent against antibiotics and host defense mechanisms. The result is increased virulence of the slime-producing strains. A number of techniques have been developed in our laboratories which facilitate identification of such bacterial pathogens on biopsy or postmortem specimens. These light and analytical electron microscopic methodologies include special cytochemical staining and rapid drying and embedding methods. Their efficacy and accuracy have been verified by studies on cultured and subcultured pathogens which are more time consuming. It is of interest that the microscopic methods showed the presence of macrophages as well as neutrophils on the specimens.

Significance of Improved Cytochemical Methods for Hemoprotein Enzymes in Diagnosis and Classification of Leukemia

Success in achieving remissions of acute myelogenous leukemia (AML) since the 1950s, and especially since the mid-1970s, has developed a need for greater attention on the part of physicians to improved methods for diagnosing and classifying the leukemias. The majority of AML patients can achieve first remissions, some (5–10%) lasting several years; these prolonged survivors are presumably cured of their leukemia. Many AML patients, however, are still not receiving the benefits of improved diagnostic methods and intensive therapeutic regimens. Of the latter group 80% are dead within a year of diagnosis [1].