A. Bolková

Biochemical study on some acid hydrolases in the normal rabbit cornea.

The activities of acid phosphatase, β-glucuronidase, β-galactosidase, and N-acetyl-β-D-glucosaminidase were investigated in the normal rabbit cornea. For all spectrophotometric assays, appropriate p-nitrophenyl derivates were used. Only β-glucuronidase was determined employing Phenolphthalein glucuronid as a substrate. Acid phosphatase revealed the highest activity, followed by N-acetyl-β-D-glucosaminidase and β-galactosidase. In the case of β-glucuronidase the lowest activity was found. The results on the rabbit cornea are compared with those on some other tissues described in the literature. Correlation of biochemical data and histochemical findings in the same species is briefly discussed. Bestimmung der Fermentaktivität der sauren Phosphatase, β-Glukuronidase, β-Galaktosidase und N-acetyl-β-D-Glukosaminidase in normalen Kaninchenhornhäuten. Für alle spektrometrischen Bestimmungen wurden die entsprechenden Substrate von den p-nitrophenyl-Derivaten angewandt. Nur β-Glukoronidase wurde mit Phenolphthaleinglukuronid nachgewiesen. Die saure Phosphatase hatte die höchste Aktivität, die N-acetyl-β-D-glukosaminidase und β-Galaktosidase folgten. Die geringste Fermentaktivität wies die β-Glukuronidase auf. Unsere Ergebnisse, die Kaninchenhornhaut betreffend, werden verglichen mit den in der Literatur angegebenen Enzymaktivitäten in anderen Geweben. Kurze Diskussion der Korrelation der biochemischen und histochemischen Befunde in derselben Spezies.

Changes of activity of alkaline and acid phosphatase in the rabbit eye in the early phase of alkaline and acid injury.

Rabbit corneas were burned either with 1.0 N sodium hydroxide or 1.0 N hydrochloric acid. Enzyme activities of alkaline and acid phosphatases were examined spectrophotometrically in the homogenates of cornea, iris, aqueous humor and vitreous body. On the 3rd day after alkaline as well as acid burn, a significant decrease of both enzyme activities was produced as compared with untreated animals. A more pronounced change was found in the case of alkaline injuries. With both kinds of caustic agents the decrease of acid phosphatase activity was more striking than that of the alkaline phosphatase. Advantages and shortcomings of biochemical and histochemical enzymatic determinations in experimental ocular inflammations are briefly discussed. Die Cornea von Kaninchenaugen wurden entweder mit 1N Natriumhydroxyd oder mit 1N Salzsäure geätzt. Spektrografische Bestimmung der Fermentaktivität der alkalischen und sauren Phosphatasen in Hornhaut, Regenbogenhaut, Kammerwasser und Glaskörper. 3 Tage nach der durch Natriumhydroxyd oder Salzsäure hervorgerufenen Schädigung zeigte sich signifikante Aktivitätsabnahme beider Fermente. Stärkere Veränderungen wurden nach Alkalischädigung gefunden. Beide kaustische Einwirkungen verursachten eine deutlichere Aktivitätsabnahme der saueren als der alkalischen Phosphatase. Kurze Diskussion der Vorteile biochemischer und histochemischer Methoden für Fermentbestimmungen bei der experimentellen Augenentzündungen.

A study on alkaline phosphatase in cornea of various animals with special regard to keratocytes.

The distribution of alkaline phosphatase was investigated in corneas of various animals by means of histochemical and biochemical methods. Special attention was paid to keratocytes that proved to be positive when a proper substrate and technique were used even if the activity differed according to animal species. Naphthol-AS-MX-phosphatate with Variamine-Blue-RT salt in the simultaneous azocoupling method was the most sensitive substrate; less suitable were naphthol-AS-phosphate, and particularly l-naphthyl-phosphate with Fast-Blue-BB-salt in the same method. Keratocytes were completely negative with β-glycerophosphate in the Gomori technique. Contrary to keratocytes, epithelial and endothelial cells were without substrate predilection. The results of both approaches showed a wide interspecies variability. The overall activity of alkaline phosphatase detected histochemically and its activity in the unsedimentable fraction of whole corneas determined biochemically was in good agreement. The highest activity was found in the calf cornea; enzyme levels decreased gradually in the bovine, rabbit, dog, sheep, and pig cornea. Die Verteilung der alkalischen Phosphatase wurde in der Hornhaut von verschiedenen Species histochemisch und biochemisch bestimmt. Besondere Beachtung fanden die Keratocyten, die man gegebenenfalls nachweisen konnte, auch wenn die Fermentaktivität verschieden war. Als empfindlichstes Substrat erwies sich Naphthol-AS-MX-Phosphat mit Variamin-Blau-RT-Salz in der simultanen Azokopulationsreaktion. Weniger geeignet waren Naphthol-AS-Phosphat, namentlich l-Naphthyl-Phosphat mit Fast-Blau-BB-Salz. Mit β-Glycerophosphat in der Technik von Gomori ließen sich keine Keratocyten nachweisen. Epithel- und Endothelzellen blieben frei von den angewandten Substanzen. Insgesamt ergaben sich erhebliche Unterschiede zwischen den einzelnen Tierarten. Die Totalaktivitäten der alkalischen Phosphatase waren gut korreliert. Die stärkste Fermentaktivität wurde in der Hornhaut von Kälberaugen gefunden, zunehmend geringer waren die Aktivitäten in der Hornhaut von Rindern, Kaninchen, Hunden, Schafen und Schweinen.

Species Variation of Some Acid Hydrolases in the Normal Cornea

Acid phosphatase, N-acetyl- β -D-glucosaminidase, β-alactosidase and β-glucuronidase were investigated in the normal corneas of several animals (rabbit, calf, bovine, dog, pig, and sh

Biochemical and histochemical response to a complete epithelial denudation of the rabbit cornea

Activities of alkaline and acid phosphatases were investigated in rabbit corneas after a complete epithelial denudation in vivo (limbus to limbus). Dynamics of enzymatic changes during corneal healing were followed quantitatively in homogenates of regenerated epithelium and stroma (denuded cornea) and in cryostate frozen sections on days 1, 4, 7, 14, and 28. Biochemical and histochemical findings at these times showed a different response in each enzyme. Acid phosphatase displayed a gradual increase of activity in epithelium as well as in stroma; on day 28 after injury its content was normal. In contrast, alkaline phosphatase showed delayed activity during the repair process, and even a month after de-epithelization was still subnormal, particularly in superficial layers of epithelium. Untersuchung der Aktivitäten der alkalischen und sauren Phosphatasen in Kaninchenhornhäuten nach totaler Abrasio in vivo. Analyse der Dynamik der Enzymänderungen während der Wundheilungsphase. Bericht über die festgestellten biochemischen und histochemischen Beobachtungen. Die saure Phosphatase zeigte eine kontinuierliche Aktivitätszunahme im Epithel und Stroma; Am 28. Tag nach der Abrasio war die Aktivität der sauren Phosphatase normal. Dagegen wurde eine Aktivitätsverminderung der alkalischen Phosphatase während der Epithelregeneration beobachtet, sogar ein Monat nach der Abrasio fiel noch eine subnormale Aktivität, besonders in den oberflächlichen Epithelschichten auf.

Effect of Fixatives on Acid Mucopolysaccharides (AMPS) in the Experimentally Hydrated Rabbit Cornea

The influence of various fixatives on the rabbit cornea hydrated in distilled water for 2 and 4 h was studied by means of staining with Alcian stains at various pH, colloidal Fe3+, Alcian blue solutions with increasing concentrations of MgCl2, and toluidine blue at pH 4.5. The initial hydration resulted in an increased intensity of staining of carboxyl groups and with the stainability of sulphate groups. These changes induced by primary hydration remained qualitatively unchanged after treatment with different fixatives. Differences were observed only in degrees of staining intensities. In comparison with other fixatives, formol-cetylpyridinium chloride (CPC) fixation significantly decreased the staining intensity of stromal AMPS.

Changes in alkaline and acid phosphatases of the rabbit cornea following experimental exposure to ethanol and acetone: A biochemical and histochemical study

Alkaline and acid phosphatases were studied in the rabbit cornea following acetone and ethanol exposure to the eye. Changes in enzyme activities were investigated in homogenates of epithelium and stroma quantitatively and in frozen cryostat sections on days 1, 4, 7, 14, and 28. Biochemical and histochemical findings showed a remarkable increase in alkaline phosphatase of the epithelium beginning on day 7. This activation persisted until day 28 after instillation of both noxae with maximum activity on day 14. However, acetone was proved to be significantly more effective than ethanol. In addition, a different topochemistry of alkaline phosphatase was found in the epithelium of treated corneas, i.e., enzyme activity was observed not only superficially but in all epithelial layers of the cornea as compared to a normal one. The effect of acetone and ethanol on a regenerating corneal epithelium is discussed.

Hydration Properties of the Corneal Stroma in Dogfish Scyliorhinus canicula L.

Cornea of the dogfish (Scyliorhinus canicula L.), with and without epithelium, were excised in solutions of different pH. The dogfish cornea was capable of swelling in acidic solutions, degree of swelling being highest at pH 3.0 from the studied range. Also some swelling might be found at alkaline pH round 12.0–13.0 followed by complete solubilization. Increased hydration of the elasmobranch cornea at various pH values was always accompanied by a remarkable proportional loss of its final dry weight. Behaviour of the elasmobranch cornea differed from properties of mammalian and teleostean corneas. The dogfish cornea was capable, contemporarily, to swell and lose its solid components

Differences in hydration characteristics of corneas in various animal species

Corneas of various animal species (monkey, dog, pig, rabbit, ox, calf and sheep) were hydrated in distilled water for 30 min, 2 and 4 hrs. At given time intervals the increase in the water content was investigated as well as the histochemical stainability of acid mucopolysaccharides. Except in the monkey cornea, hydration caused a higher stainability not only of carboxyl groups, but also the stainability of sulphate groups, as compared with unhydrated control corneas. The highest staining intensity was observed after hydration for 2 hrs. Prolonged hydration (4 hrs) did not increase stainability, but on the contrary, it decrease a little due to loosening of the tissue. Differences among the various species lay only in the intensity of staining at the same time intervals of hydration: the lowest intensity was found in the dog cornea and it gradually increased in the pig, rabbit, bovine, calf and sheep corneas. The monkey cornea behaved differently. In this species hydration caused no increase in stainability of either carboxyl or sulphate groups as compared to controls. This fact is probably connected with a more interwoven fascicular structure of monkey cornea which limits hydration. The acidic groups of mucopolysaccharides are thus less accessible to cation dyes. Hornhäute verschiedener Tierarten (Affe, Hund, Schwein, Kaninchen, Rind, Kalb und Schaf) wurden in destilliertem Wasser für 30 min, 2 und 4 Std hydriert. Der Gewichtszuwachs wurde in den einzelnen Zeitabschnitten sowie das histochemische Bild der sauren Mucopolysaccharide verfolgt. Mit Ausnahme der Affen-Cornea bewirkte Hydratation nicht nur eine höhere Farbfähigkeit der Carboxylgruppen, sondern auch die Farbfähigkeit der Sulfatgruppen. Die stärkste Intensität der Färbung wurde nach 2 Std der Hydratation gefunden. Der folgende Zeitabschnitt (4 Std) bewirkte schon keine Intensivierung der Färbung. Der Unterschied zwischen den einzelnen Tierarten bestand nur im Grad der Farbfähigkeit innerhalb desselben Zeitabschnittes; die geringste Farbfähigkeit wurde beim Hund und allmählich zunehmend beim Schwein, Kaninchen, Rind, Kalb und Schaf beobachtet. Ganz anders verhielt sich die Affen-Cornea: die Farbfähigkeit derselben erhöhte sich nicht bei Hydratation, weder für die Carboxyl-, noch für die Sulfatgruppen.

Acid Mucopolysaccharides in Non-Swelling Dogfish Corneas

The non-swelling properties of dogfish corneas were studied using histochemical and biochemical methods for estimating acid mucopolysaccharides. Immersion of the corneas in distilled water caused loss