Arto Palkama

Improved localization of phosphorylase by the use of polyvinyl pyrrolidone and high substrate concentration.

585 Truks’uschui romoob 1itsmiroki (.1. Hislochem. (]ylochem. 3: 153, 1955) s!s’ns’mihues! a sois’thod for histochenouical ohesoionsslmrotions of phosphom)’lase. This moset-hod is thus’oretictohby notososh amusl ovorks ins practice. Howeven, ooit-hu tinsussri less roctive, for instance, than liven titus! susumncle, t hue suet hos! I)movs’51 somuuewhat ctui)mis’isuuos in siuom hunumotlri asis! toe decisled to investigate the effect of riotous’ factors involved ins the ns’ros’tioss ins thus’ hoops’ to imOupmovs’ the nsiethooh. The suunsiss resuslts time enlists’s! us Table I. The main diffs’rens’es his’tween the above proceslurs’ and that given by Taks’uchi and Kumiaki rums’ the presence of I)ol)’visuyh pyrrohidone tons! I-he higher concs’mstmabiomo of glucose-I-phosphate ins the substrate nuixtusme. Both of these chtomugs’s significantly iniprovesl the sensitivity ansI ths’ localizing power of the reaction. The advamutageoums effect of l)olyvinYl pymmohidone has bes’ss ohsemves! earlier in dehys!mogemsrose techniquss’s ansi it os-as ascnil)s’d to osmsuobic protection (Scarpelhi rosusi

Fine structure of sensory nerves in the rat cornea: an experimental nerve degeneration study.

&NA; Fine structure of nerve fibres and terminals in the rat cornea was studied after fixation with glutaraldehyde and osmium tetroxide or with potassium permanganate. Superior cervical and/or ciliary ganglionectomy as well as ophthalmic neurotomy were performed in order to verify the origin of the corneal nerves. In the control corneas axon profiles with granular vesicles were observed in the stroma. Other axons containing agranular vesicles were found both in the stroma and in the epithelium. Superior cervical ganglionectomy abolished only the axons containing granular vesicles but did not affect the corneal blink reflex. Ciliary ganglionectomy induced more conspicuous changes in both stromal and intraepithelial axon profiles. The corneal reflex was, however, normal. Some of the axons containing agranular vesicles also showed degenerative changes. However, intact axon profiles with agranular vesicles were seen even after extirpation of both the ciliary ganglion and the superior cervical ganglion (combined autonomic ganglionectomy). On the other hand, all axon profiles with agranular vesicles disappeared after coagulation of the ophthalmic and maxillary nerves (sensory denervation). This operation destroyed most of the axons in all parts of the cornea and abolished the blink reflex. It is concluded that the axon profiles in the corneal epithelium, containing agranular vesicles, are sensory axons of the trigeminal nerve. A possible role of the vesicles in sensory nerves is discussed.

Calcitonin gene-related peptide (CGRP) immunoreactive sensory nerves in the human and guinea pig uvea and cornea

The presence of calcitonin gene-related peptide (CGRP) immunoreactive nerves in the uvea and cornea of human and guinea pig eyes was evaluated using immunohistochemical techniques. CGRP immunoreactivity was found in thin, varicose nerve fibers in both species. Most of the fibres were localized in the ciliary body, and were mainly associated with blood vessels. In the human ciliary body, a moderate number of CGRP immunoreactive nerves were also seen in the ciliary muscle. In the iris and cornea, CGRP immunoreactive fibres were relatively uncommon. In the iris, they were mostly found associated with blood vessels, while in the cornea they were seen sub-epithelially or as free nerve endings in the epithelium. In the trigeminal ganglion, small sized ganglion cells displayed CGRP immunoreactivity. About 40% of all ganglion cells were immunoreactive nerves in the guinea pig, while sympathetic denervation did not change the staining pattern of CGRP immunoreactivity. The present findings, together with previous physiological data, suggest that CGRP might play a role in the regulation of the blood flow, aqueous humour dynamics, and neurogenic inflammation, not only in experimental animals but also in man.

An electron microscopical study of the blood-aqueous barrier in the ciliary body and iris of the rabbit☆

Abstract Horseradish peroxidase was administered to rabbits by intravenous injection, and its distribution in the iris and in the different regions of the ciliary body was studied using an electron microscope. In the iris, exogenous peroxidase was localized in the lumina of the blood vessels and in some micropinocytotic vesicles within the endothelial cells. None was found beyond the vascular endothelium. The micropinocytotic vesicles were few in number and did not appear to transport peroxidase in great amounts, while the tight junctions between the endothelial cells were probably responsible for preventing its intercellular passage. These findings indicate the presence of a “barrier” at the endothelium of the iris vessels. In the different regions of the ciliary body exogenous peroxidase was localized in the lumen of the vessels, in the vesicles throughout the cytoplasm, and in the intercellular clefts between the endothelial cells. Transport may occur through fenestrations rather than through the cytoplasm of the endothelial cells. Peroxidase easily penetrated the stroma and also the basement membrane of the vessels and pigmented cells and the intercellular spaces between the pigmented cells. The ultrastructural observations indicated that the non-pigmented epithelial cells and their intercellular junctions constitute the site of the barrier to proteins under normal conditions. Only in the ciliary processes and the pars plana was the reaction product seen to penetrate into the intercellular spaces between two adjoining non-pigmented cells. The significance of the different permeabilities in the junctions of the non-pigmented epithelial cells in different regions of the ciliary body is discussed, particularly with reference to histochemical and cytochemical differences.

Effect of neurogenic irritation and calcitonin gene-related peptide (CGRP) on ocular blood flow in the rabbit

The effects of sensory nerve stimulation (topical neutral formaldehyde, 1%) and intracameral injection of calcitonin gene-related peptide (CGRP) on regional ocular blood flow, intraocular pressure (IOP), the blood-aqueous barrier, pupil size, and blood pressure were studied in the rabbit. Sensory nerve stimulation elicited a typical irritative response in the rabbit eye, with vasodilation in the ciliary body (from 128 +/- 31 to 363 +/- 105 mg/min, p less than 0.05) accompanied with a breakdown of the blood-aqueous barrier, rise in the IOP, and miosis. CGRP caused similar, but not identical, changes in the eye: vasodilation in the ciliary body (from 60 +/- 14 to 258 +/- 75 mg/min, p less than 0.05), breakdown of the blood-aqueous barrier and rise in the IOP, accompanied with systemic hypotension. Miosis was not observed after CGRP. In the present study, the vasodilatory action of CGRP on the rabbit eye has been shown. This makes our understanding of the mechanism of the ocular irritative response after sensory nerve stimulation more complete. Thus, CGRP through vasodilation disrupts the blood-aqueous barrier and raises the IOP. The more intense increase in the IOP after sensory nerve stimulation than after CGRP is probably caused by a CGRP-induced vasodilation and breakdown of the blood-aqueous barrier, enhanced by a miosis-induced pupillary block.

Electronmicroscopical, histochemical and biochemical findings on the Na-K-ATPase activity in the epithelium of the rat lens

Abstract The sodium-potassium activated adenosine triphosphatase (Na-K-ATPase) activity in the epithelium of the rat lens was investigated histochemically using light and electron microscopy and biochemistry. The histochemical demonstration of Na-K-ATPase activity in the lens epithelium was modified to be highly specific and to give a precise localization. The enzyme reaction was localized on the epithelial cell membranes that are adjacent to neighbouring epithelial cell membranes, whereas the apical and basal cell membranes were negative. The reaction product was almost totally absent in the presence of the specific enzyme inhibitor (ouabain). The reaction was strongly decreased also in the absence of the specific enzyme activators (sodium and potassium). The lens capsule as well as the lens fibres were negative. Biochemical studies of the kinetic properties of the lens epithelium were characterized by studying the effect of various chemicals on Na-K-ATPase activity. The enzyme activity was increased by the Na + , K + and Mg 2+ and inhibited by ouabain. Enzyme activity was optimal when Mg 2+ ATP ratio was approximately 1·4 and the pH at 7·7. Optimal activity was also obtained by using 85·0 m m -sodium and 1·0 m m -potassium in the solution. The results in this study indicate that the adjoining cell membranes of the lens epithelium contain the Na-K-ATPase activity and that these are probably responsible for the exchange of water and ions across the lens epithelium.

Penetration barrier to sodium fluorescein and fluorescein-labelled dextrans of various molecular sizes in brain capillaries

The permeability of the blood-brain barrier to sodium fluorescein, or fluorescein-labelled dextrans of various molecular weights, was investigated. Unlike the capillaries in both the area postrema and the eminentia mediana, the capillaries of the cerebral cortex were impermeable to all the intravenous tracer substances used.

Distribution of adrenergic nerves in the lacrimal glands of guinea-pig and rat

The distribution of adrenergic nerves in the ex- and intraorbital lacrimal glands of guinea-pig and rat was studied using the sucrose-potassium phosphate-glyoxylic acid (SPG) technique. Blood vessels and secretory acini of guinea-pig lacrimal glands were demonstrated as having a rich adrenergic innervation. Adrenergic fibers in the rat were, however, much more sparse, and most of them were seen in association with glandular blood vessels, with only a few being found between secretory acini. Pretreatment of animal with a monoamine oxidase inhibitor and L-dopa did not change the morphological distribution of catecholamine fluorescent fibers, although the treatment improved the fluorescence, especially in the rat. Extirpation of the ipsilateral superior cervical ganglion eliminated all the fluorescent fibers in both normal and pretreated animals. The presence of adrenergic innervation of the lacrimal glands, especially in close connection with secretory acini, supports the theory that catecholamine-containing nerves play a role in the regulation of lacrimal secretion.

Biochemical and immunohistochemical determination of 5-hydroxytryptamine located in mast cells in the trigeminal ganglion of the rat and guinea pig

SummaryThe presence of 5-hydroxytryptamine (5-HT), as well as its precursor (5-HTP) and metabolite (5-HIAA), were biochemically determinated in the trigeminal ganglion of the guinea pig and rat. The distribution of 5-HT in the ganglion and in its posterior root was studied using both indirect immunofluorescence and the peroxidase-antiperoxidase method. In order to increase the possible 5-HT content of primary sensory neurons for subsequent immunohistochemical visualization, animals were first treated with nialamide, an inhibitor of monoamine oxidase, and then loaded with l-tryptophan. Another group of animals received colchicine to inhibit intra-axonal transport of transmitter substances. However, even combined use of loading and colchicine treatment did not reveal 5-HT immunoreactivity in ganglion cells.The only source of 5-HT immunoreactivity in the trigeminal ganglion and its posterior root was mast cells. These cells were located around the ganglion in adjacent leptomeningeal and connective tissues, as well as between the ganglion cells and nerve fibers. Only occasionally were mast cells found in the posterior root of the ganglion.

Innervation of the rabbit cornea

The innervation of the rabbit cornea was investigated histochemically and electron-microscopically with.special reference to the autonomic nerves. Both formaldehyde- and glyoxylic-acid-induced fluores