A quantitative histochemical study of NADPH-ferrihemoprotein reductase activity

Summary A quantitative histochemical assay for NADPH-ferrihemoprotein (P450) reductase had been developed. For optimal activity, it is necessary to use a relatively electropositive tetrazolium salt such as neotetrazolium chloride as the final acceptor. The apparentK _m of the reaction is 0.83 mM. Its specificity has been proven in two ways: (i) activity is increased selectively in the pericentral zone of liver from rats treated with phenobarbitone, an inducer of the reductase, though not in liver of rats injected with 3-methylcholanthrene, which induces NAD(P)H dehydrogenase; (ii) it is competitively inhibited by NADP⁺ (K _i=1.50mm) though unaffected by dicumarol, an inhibitor of NAD(P)H dehydrogenase activity. An NADP⁺ concentration ten times greater than the substrate concentration inhibits the histochemical reaction and the reaction in a microsomal fraction assayed biochemically to the same degree (70% inhibition). The amount of inhibition is independent of temperature, of the zone of the acinus and of the treatment of the animal.Continuous microdensitometric monitoring of the reaction product as it is formed has shown that the specific reaction is linear with incubation up to 10 min, thus allowing end-point measurements to be used for cytophotometric analysis.     

A quantitative study of changes in the human corpus luteum microvasculature during the menstrual cycle

Endothelial cells are the most abundant cell type in the corpus luteum (CL), and changes in blood vessels have been proposed to play a pivotal role in CL regression. We have studied quantitatively the changes in the human granulosa-luteal microvasculature in CL of various ages: young (Days 17-19 of the cycle), mature (Days 20-24), old (Days 25-27), early regressing (follicular phase of the following cycle), and late regressing (luteal phase of the following cycle). Blood vessels were identified by immunohistochemical staining for the endothelial cell marker CD34. Because of the anisotropy of blood vessels, both vertical and transverse sections of the granulosa-lutein layer (GLL) were used to estimate relative (volume, surface, and length densities) and absolute (mean cross-sectional area) vascular variables. Full luteinization from young to mature CL was accompanied by a 61% increase in the mean cross-sectional area of vascular profiles and a 52% increase in the mean volume of granulosa-lutein cells, as an estimator of changes in the volume of the GLL. In old and early regressing CL, there was a progressive increase in relative structural vascular variables, due to the shrinkage of the GLL, whereas the mean cross-sectional area of capillaries showed a 53% decrease from mature to old CL. Finally, in late regressing CL, there was a decrease in most relative structural variables, in spite of the increasingly shrunken GLL. The decrease in the capillary diameter found at the late luteal phase most likely leads to a decreased blood flow, and early changes in blood vessels could initiate and/or accelerate CL regression.     

A quantitative histochemical study of dipeptidylpeptidase IV (DPP IV)

Summary In order to elucidate the possibility of a quantitative study of dipeptidylpeptidase IV (DPP IV) in cells and cell compartments of tissue sections kinetic investigations were performed with biochemical fluorometric and cytophotometric (microdensitometric) methods in the jejunum, kidney and liver of adult rats; in addition, two approaches of microdensitometric measuring (endpoint and continuous cytophotometry) were compared. Biochemically, Gly-Pro-4-methoxy-2-naphthylamine (MNA) is the best substrate compared with the 1-naphthylamine and 2-naphthylamine due to its high hydrolysis rate, the low Michaelis constant (K_m) and the relative high fluorescence of MNA. Its pH optimum is between 8.5 and 9. The hydrolysis rates delivered by cacodylate, phosphate and Tris HCl are similar and always higher than with other buffers. The maximal reaction velocity is reached with 3 mM Gly-Pro-MNA. The hydrolysis is inhibited in the presence of phenantroline, diisopronyl fluorphosphate, formaldehyde and diazonium salts; furthermore, formaldehyde and diazonium salts increase the Km of DPP IV. Fast Blue B pure (FBB) is the simultaneous coupling reagent of choice. End-point (plug and scanning procedure) and continuous microdensitometry with Gly-Pro-MNA and FBB in the intestinal and renal brush border and in renal glomerula have to be carried out at suboptimal pH (7.5). However, the optimal substrate concentrations are identical and the overall relation between the activity of DPP IV in the jenum, liver and kidney are similar in the quantitative histochemical and biochemical system. A linear relationship between the quantity of azo-dye and section thickness or incubation time exists between 4 m and 10 m and during the first 6 min of incubation respectively. Among different plotting procedures the best-fit method delivers the most reliable results. — The microdensitometric data in the small intestine show that K_m and V_max differ at different positions of the villi and may represent parameters for the maturation process of the enterocytes; in the kidney microdensitometry reveals different DPP IV activities in the different parts of the nephron. Both, the findings for the villi and the nephron cannot be obtained by biochemical methods.Supported by Deutsche Forschungsgemeinschaft (GU 184/1)Supported in part by Deutsche Forschungsgemeinschaft (SFB 105)     

A quantitative study of the validity of the histochemical demonstration for pyridine nucleotide-linked dehydrogenases

Summary A quantitative histochemical and biochemical study has been made of the loss of pyridine nucleotide-linked dehydrogenases from frozen histological sections of rat liver. Glucose-6-phosphate, 6-phosphogluconate and lactate dehydrogenases were lost rapidly from the sections during incubation in the histochemical medium, but -OH-butyrate dehydrogenase was lost at a much slower rate. It was shown that a dehydrogenase reaction can occur in a section lacking that particular dehydrogenase if the section is incubated in the presence of another containing the dehydrogenase. The validity of the tetrazolium reaction for demonstrating pyridine-nucleotide-linked dehydrogenases is considered in the light of these results.     

Genetic heterogeneity of skin microvasculature

Angiogenesis, the formation of new blood vessels from existing vasculature, is a complex process that is essential for normal embryonic development. Current models for experimental evaluation of angiogenesis often use tissue from large vessels like the aorta and umbilical vein, which are phenotypically distinct from microvasculature. We demonstrate that the utilization of skin to measure microvascular angiogenesis in embryonic and adult tissues is an efficient way to quantify microvasculature angiogenesis. We validate this approach and demonstrate its added value by showing significant differences in angiogenesis in monogenic and polygenic mouse models. We discovered that the pattern of angiogenic response among inbred mouse strains in this ex vivo assay differs from the strain distributions of previous in vivo angiogenesis assays. The difference between the ex vivo and in vivo assays may be related to systemic factors present in whole animals. Expression analysis of cultured skin biopsies from strains of mice with opposing angiogenic response was performed to identify pathways that contribute to differential angiogenic response. Increased expression of negative regulators of angiogenesis in C57Bl/6J mice was associated with lower growth rates.     

A quantitative histochemical study of sulphydryl and disulphide content during normal epidermal keratinization

Summary A quantitative histochemical study was carried out on the distribution of protein thiol and disulphide groups in normal human plantar epidermal tissue. Histochemical demonstration of reactive groups was achieved by addition ofN-(4-aminophenyl) maleimide, subsequent diazotization and final coupling with a Nitro Red or chromotropic acid label as first described by Sippel. The quantitative reliability of the method was tested by absorption cytophotometry, and evaluated on the basis of the internal consistency of the results reported.Our histological observations and histophotometric data support accepted views on epidermal keratinization. A limited, though reproducible, amount of disulphide bonds was observed near the basement membrane. The free thiol concentration in basal and prickle cells was low and almost constant, but was higher in the granular cells, where deposition of sulphur-containing proteins on cell membranes is initiated. In Malpighian layers, disulphide cross-links only occurred just beneath the transition zone in thickened cell membranes. The staining pattern of the inner stratum corneum resembled a mosaic and was characterized by a sharp rise of the disulphide content, which exceeded the decrease in free thiol groups. The free thiol concentration decreased further throughout the cornified layers whilst the disulphide content remained fairly constant. Staining of thiol and disulphide groups together corresponded, within the limits of the standard error, to the sum of the thiol and disulphide concentrations when they were assayed separately in living and horny cells. These results confirm that living cells are the main site of free thiol groups, while horny cells are the most prominent site of disulphide cross-links.     

Reinnervation of human skin grafts: a histochemical study

The reinnervation of nine human skin grafts was investigated using histochemical thiocholine methods for the demonstration of cholinesterases. The regenerated cutaneous nerves showed both specific acetylcholinesterase and nonspecific cholinesterase reactions. In the youngest specimens, taken 3 weeks after the grafting, such regenerated nerves were seen both at the subdermal level under the graft and at the margins of the graft. These nerves seemed to orient toward the denervated graft area. The growing nerves were generally distributed in a random fashion. The reinnervation of some hair follicles, erector pili muscles, and sweat glands were observed in well-innervated full-thickness and thick partial-thickness skin grafts. It is suggested that this target-organ control of regenerating nerves occurs as a result of the action of chemotactic factors. A well-innervated graft bed seems to be important for optimum reinnervation of skin grafts. Fibrosis and scarring seem to hamper nerve regeneration.     

A histochemical study on the snout tentacles and snout skin of bristlenose catfish Ancistrus triradiatus

Histological sections of the tentacles of Ancistrus triradiatus revealed that they contain many goblet cells with granule-like sections distributed along the edge of the tentacles (mean 302 cells mm⁻¹). Various histochemical methods were used to characterize the contents of the goblet cells. The results indicated that the contents were periodic acid-Schiff (PAS) positive, glycogen negative and no sulphated mucins could be found. The contents of the goblet cells were acid mucins and O-acetylated sialomucins which had high energy content. The snout skin of the male also contained goblet cells with higher density (mean 755 cells mm⁻¹) than that of the tentacles. Snout skin of female A. triradiatus , on the contrary, did not contain any goblet cells. Instead, many minute spines could be found. Based on the acid mucins and O-acetylated sialomucins secretions of goblet cells which are of high nutritional values, it is hypothesized that snout tentacle secretions are used to provide nutrients to fish larvae when they are under paternal care.     

A quantitative microspectrophotometric study of the lead precipitation reaction for the histochemical demonstration of acid phosphatase.

The possibility of quantitating microspectrophotometrically the lead precipitation reaction for the histochemical demonstration of acid phosphatase was studied using the following models: sections of rat kidney and human prostate gland, and polyacrylamide films with incorporated prostate gland homogenate. The activity of the polyacrylamide models was previously evaluated by a biochemical method and the specificity of the reaction was controlled with specific inhibitors. Linear relations between cytophotometric reading and enzymatic activity, and between optical densities and different incubation periods, were demonstrated. The results obtained indicate the validity of the microspectrophotometric quantitation for this reaction with direct readings over a wide range of the visible light spectrum.     

A quantitative histochemical study of three oxidative enzymes in solar keratoses and Bowen's disease

Synopsis Succinate dehydrogenase (SDH), glucose-6-phosphate dehydrogenase (G6PDH) and lactate dehydrogenase (LDH) activities have been studied using quantitative enzyme histochemical techniques in the epidermis of five patients with solar keratoses and Bowen's disease. Non sun-exposed buttock skin was compared with the skin from the actual lesion and adjacent, clinically normal paralesional skin. SDH activity was significantly increased in the basal layer and decreased in the granular layer in the epidermis of both lesion and paralesional skin, although the total epidermal activities were unchanged when compared to non-exposed buttock skin. G6PDH activity was increased in the granular layer of paralesional epidermis and of lesions. No change in LDH activity was detected. Inclusion of phenazine methosulphate in the reaction mixtures resulted in a three-fold increase in formazan deposition without altering the localization.It is concluded that the quantitative changes and alteration in localization of SDH and G6PDH reported in solar keratoses are accompanied by similar changes in adjacent, clinically normal sun-exposed skin and differ from normal non-exposed skin. It is suggested that these changes may precede the development of the solar keratoses and that these findings may indicate a significant metabolic alteration in the events that lead to neoplasia.     

Diurnal variation in 5'-nucleotidase activity in rat liver. A quantitative histochemical study

The diurnal variation of 5'-nucleotidase activity in periportal and pericentral areas of rat liver parenchyma has been determined with quantitative histochemical means. 5'-Nucleotidase activity was estimated using microdensitometry in cryostat sections after being incubated with a medium according to Wachstein and Meisel (1957). It appeared that 5'-nucleotidase activity was significantly higher in pericentral areas than in periportal areas throughout the daily cycle and showed a maximum at the end of the light period. It was concluded that 5'-nucleotidase activity may be related with the capacity to diminish messenger RNA resulting in protein breakdown.     

Diurnal variation in glycogen phosphorylase activity in rat liver. A quantitative histochemical study

The diurnal variations of the glycogen content and of glycogen phosphorylase activity in periportal and pericentral areas of rat liver parenchyma have been analyzed in periodic acid Schiff (PAS)-stained cryostat sections using quantitative microdensitometry. Glycogen content and phosphorylase activity were always higher in periportal areas than in pericentral areas throughout the daily cycle. The glycogen content was highest at the end of the active period during darkness and lowest at the end of the resting period. Phosphorylase activity appeared to be inversely correlated with the glycogen content in both areas. It is concluded that the glycogen content is regulated by phosphorylase activity, which may be due to local cAMP concentration.