Immunohistochemical demonstration of neurotensin and tyrosine hydroxylase in iris nerves of the rabbit eye

In previous studies we have provided evidence that intracameral administration of neurotensin (NT), an endogenous tridecapeptide, produces strong miosis in the rabbit. The presence of NT immunoreactivity was investigated in rabbit iris whole mounts by light microscopic immunohistochemistry, and its distribution in the iris compared to that, of tyrosine hydroxylase (TH). A few scattered NT-positive cell bodies were localized in the dilator muscle. Both, the NT cell bodies and processes appeared parallel to the muscle cells. Extensive branching of NT-containing cell processes was observed in connection with the sphincter muscle. These NT-positive fibers formed a dense, randomly oriented network throughout the sphincter muscle cells. The distribution of TH immunoreactivity was similar to that of NT-positive cell processes, except that no TH-positive cell bodies were detected in any of the iris structures examined. Moderate branching of TH-positive fibers was observed in the dilator and sphincter iris muscles. These findings provide neuroanatomical support for an important role of NT in pupillary physiology. Its similar topographical distribution with TH suggests that NT and dopamine may be co-localized, as it has already been described in brain.     

Immunohistochemical demonstration of neurotensin and tyrosine hydroxylase in iris nerves of the rabbit eye

Summary In previous studies we have provided evidence that intracameral administration of neurotensin (NT), an endogenous tridecapeptide, produces strong miosis in the rabbit. The presence of NT immunoreactivity was investigated in rabbit iris whole mounts by light microscopic immunohistochemistry, and its distribution in the iris compared to that, of tyrosine hydroxylase (TH). A few scattered NT-positive cell bodies were localized in the dilator muscle. Both, the NT cell bodies and processes appeared parallel to the muscle cells. Extensive branching of NT-containing cell processes was observed in connection with the sphincter muscle. These NT-positive fibers formed a dense, randomly oriented network throughout the sphincter muscle cells. The distribution of TH immunoreactivity was similar to that of NT-positive cell processes, except that no TH-positive cell bodies were detected in any of the iris structures examined. Moderate branching of TH-positive fibers was observed in the dilator and sphincter iris muscles. These findings provide neuroanatomical support for an important role of NT in pupillary physiology. Its similar topographical distribution with TH suggests that NT and dopamine may be co-localized, as it has already been described in brain.     

Catecholamines in adrenergic nerve fibers of the lymph nodes after sympathectomy

Using Falck method in combination with microfluorimetry, catecholamine level in adrenergic nervous fibers has been measured in the canine popliteal lymph nodes, normal and in 12 h, 7, 30, and 90 days after unilateral lumbar sympathectomy. During first 24 h after the operation the level of catecholamines is for certain increased in the nervous fibers of the lymph node of the sympathectomized extremity. In 30 days after the sympathectomy their content drops at the side of the operation and increases in the contralateral extremity. By 3 months the equilibrium of the catecholamine content is restored in the nervous fibers of the lymph nodes in the homo- and contralateral extremities at the level higher than in the control. A conclusion is made that under conditions of unilateral sympathectomy only a partial sympathectomy of the popliteal lymph node is reached. All luminiscent adrenergic nervous fibers of the sympathectomized lymph node are processes of neurons, situating in the contralateral sympathetic trunk, or neurites of cells in sacral nodes, getting their preganglionic fibers from the contralateral trunk. The changes in catecholamine concentrations mentioned are considered as a compensatory reaction, directed to maintenance of general homeostatic equilibrium under conditions, when the nervous system transfers to a new level, ensuring the partly desympathized tissue by mediators.     

Ultrastructural changes in the nerves innervating the cerebral artery after sympathectomy

Summary The ultrastructure of the innervation of the anterior cerebral artery of the rat was studied in control animals and in animals after superior cervical ganglionectomy.Fluorescence histochemistry shows a periarterial network of intensely fluorescent fibers which are divided into two groups, adventitial and periadventitial. The fluorescence begins to decrease 26 hours after, and completely disappears about 32 hours after, ganglionectomy.Fine structural changes are first observed 18 hours after ganglionectomy, when the axoplasm of degenerating axons becomes electron dense. This density gradually increases up to about 32 hours. By 32 hours most axons with disintegrating axolemmas become inclusion bodies of the Schwann cells. At this stage, synaptic vesicles can still be distinguished as less dense areas, but the membrane structures of synaptic vesicles and mitochondria are difficult to recognize. The degenerating axons are gradually absorbed and by 38 hours dense, residual bodies are observed in the Schwann cells. Generally speaking, the degeneration occurs first in the adventitial fibers and then in the periadventitial fibers. The transient appearance of small, granular vesicles is noticed in axon terminals about 18 hours after denervation, although very few small, granular vesicles are seen in control tissue or at later stages of degeneration.     

Histochemistry and ultrastructure of adrenergic and acetylcholinesterase-containing nerves supplying follicles and endocrine cells in the guinea-pig ovary

Summary With the use of an anti-human S-100 protein antibody, it was possible to reveal a characteristic cell type in the anterior lobe of the normal human pituitary. These cells, so-called folliculo-stellate cells, were present in all pituitaries studied but their number varied from one gland to another. Immunoreactive cells, isolated or grouped, were arranged close to various secretory granulated cells. Especially by use of double immunoenzymatic labeling, it was evident that these cells are spatially related either to somatotropes, prolactin cells and corticotropes, or to glycoprotein-containing cells. Such immunoreactive cells were rare or absent in pseudo-follicular arrangements of secretory granulated cells. Since it is now possible to identify this cell type by light microscopy and since no reliable functional significance is known, it seems more advisable to term this cell type stellate cell instead of folliculostellate cell.     

Changes in the myocardial ultrastructure of the perfused rabbit heart under hypoxia

Ultrastructure of rabbit heart left ventricle isolated according to Langendorf was examined under different conditions: in intact animals, during ligation of the coronary artery, and hypoxic heart perfusion. The intact perfused heart showed unmarked exo- and intracellular edema and moderate swelling of the mitochondria. During hypoxic perfusion, marked swelling and destruction of the mitochondria were noted. During ligation of the coronary artery, the heart was characterized by a high degree of mitochondrial heterogenicity. The correlation of the data obtained allowed one to reveal the myocardial adaptive-accommodative mechanism, (intermittent mitochondrial activity) that makes it possible to maintain the heart bioenergetics during coronary artery occlusion at a permanently high level.     

Survival and ultrastructure of gene-microinjected rabbit embryos after vitrification

Morphological signs of injury and subsequent regeneration following vitrification of either rabbit gene microinjected (Gene-Mi) or intact in vitro cultured embryos derived from in vivo fertilized eggs were evaluated by post-warming recovery in culture and analysed by transmission electron microscopy (TEM). The percentages of vitrified/warmed Gene-Mi embryos that reached the blastocyst stage (69%) and hatched (57%) did not differ significantly from those of intact embryos (78% and 56%, respectively). In contrast, in vitro development of embryos to the blastocyst stage among non-vitrified intact (96%) and Gene-Mi (90%) embryos compared with both the intact vitrified (78%) and Gene-Mi vitrified (69%) groups, as well as hatching rate (94%, 90% vs 56%, 57%, respectively) varied significantly (p < 0.001). Observations by TEM showed that the vitrified/warmed intact or Gene-Mi embryos without post-culture displayed severe degenerative changes among their cells. During 24 h of culture a proportion of the embryos were able to regenerate and complete the compaction process. Nevertheless the signs of previous injury were retained, such as swollen cytoplasmic organelles and remaining cellular debris in the perivitelline space. These observations indicate that the procedure of gene Mi does not significantly compromise embryo tolerance to cryopreservation and post-warming developmental ability. Severe changes in embryo morphology, observed at the ultrastructural level, can be attributed to a direct influence of the vitrification process rather than to the Mi procedure itself.     

Effects of the parkinsonism-inducing neurotoxin MPTP and its metabolite MPP+ on sympathetic adrenergic nerves in mouse iris and atrium

The effect of systemic administration of the parkinsonism-inducing neurotoxin MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) and its metabolite MPP+ (1-methyl-4-phenylpyridine) on sympathetic adrenergic nerves in mouse iris and atrium has been investigated employing histo- and neurochemical techniques. The results indicate that MPTP does not have any potent neurotoxic effects on sympathetic adrenergic nerves. The effects of MPTP noted appear mainly to be restricted to a noradrenaline (NA) -depleting action and an acutely transient impairment of the NA uptake mechanism. This latter effect could be counteracted by monoamine oxidase inhibition. MPP+ was found to have more potent neurotoxic actions than MPTP as reflected i.e. by a patchy loss of histochemically demonstrable adrenergic nerves in iris which persisted for at least 7 days. Pretreatment with the NA uptake blocker desipramine antagonised the effects of MPP+, indicating that neurotoxicity is mediated via the NA uptake mechanism. The difference in neurotoxic potency of MPTP between sympathetic adrenergic nerves and central catecholamine neurons might be related to differences in metabolism of MPTP in the CNS and the periphery and/or due to the sympathetic adrenergic nerves being more resistant towards the cytotoxic actions following MPTP administration.     

The synthesis of NPY and DBH is independently regulated in adrenergic nerves after reserpine

A newly developed cytofluorimetric scanning technique was applied in a pharmacological study to investigate the influence of reserpine (10 mg/kg) on the axonal transport of norepinephrine (NE), dopamine--hydroxylase (DBH), tyrosine hydroxylase (TH), and neuropeptide Y (NPY)-like immunoreactivities (LI) in the adrenergic axons of the sciatic nerve of rat. Early after reserpine (18 hr and 24 hr after the reserpine injection) the amounts of NE accumulated proximal to a 12-hr crush werenil or very low, as observed in earlier studies. DBH-LI, TH-LI, and NPY-LI accumulations were also depressed but only to about 50% of control accumulations. This decrease in amounts of transported substances was probably caused by a decrease in protein synthesis and also a lowered velocity of fast axonal transport initially after reserpine, when body temperature is low. The amounts of accumulated NE, DBH-LI, TH-LI, and NPY-LI were normalized around day 2 after reserpine, but on day 4 NE, DBH-LI, and in some rats also TH-LI accumulated in supranormal amounts. However, NPY-LI accumulations were normal, indicating that DBH, butrot NPY, was trans- synaptically induced in rat sympathetic neurons, and that the biochemical composition of axonally transported organelles is altered for some days after reserpine.Dedicated to Dr. Abel Lajtha.     

Chronic hypoxia alters prejunctional alpha(2)-receptor function in vascular adrenergic nerves of adult and fetal sheep

The impact of development and chronic high-altitude hypoxia on the function of prejunctional alpha(2)-adrenoceptors was studied by measuring norepinephrine release in vitro from fetal and adult sheep middle cerebral and facial arteries. Blockade of prejunctional alpha(2)-adrenoceptors with idazoxan significantly increased stimulation-evoked norepinephrine release in normoxic arteries. This effect was eliminated after chronic hypoxia in cerebral arteries, with a tendency to decline in fetal facial arteries. After chronic hypoxia, the capacity to release norepinephrine declined in fetal middle cerebral arteries with a similar trend in facial arteries. Norepinephrine release was maintained in adult arteries. During development, stimulation-evoked norepinephrine release from middle cerebral and facial arteries was higher compared with adult arteries. In fetal arteries, adrenergic nerve function declined after chronic hypoxia. However, in adult arteries, adrenergic nerves adapted to chronic hypoxia by maintaining overall function. This differential adaptation of adrenergic nerves in fetal arteries may reflect differences in fetal distribution of blood flow in response to chronic hypoxic stress.     

Fine structure and fluorescence morphology of adrenergic nerves after 6-hydroxydopamine in vivo and in vitro

Summary In parallel fine structural, fluorescence histochemical and biochemical experiments the effect of 6-OH-DA administered in vivo and in vitro on the adrenergic nerves in the mouse iris was studied. As seen in the electron microscope, in vivo administration of 6-OH-DA causes a selective, rapid degeneration of the adrenergic axon terminals similar to that found after axotomy, whereas the cholinergic nerves are unaffected at all time intervals studied. Already 1 hr after the injection of 6-OH-DA the axonal enlargements swell and the size of the dense core of the granular vesicles is strongly reduced. Since the NA stores are almost completely depleted at this time interval, the small core present may be due to a reaction between 6-OH-DA and the fixative. From 2–4 hr after the injection increasing numbers of axonal enlargements with a high electron density are observed in the Schwann cell cytoplasm, which later are digested and completely absent about 48–72 hr after the 6-OH-DA injection. During the following weeks adrenergic axons reappear. This time course of degeneration obtained is considerably faster than that seen after axotomy in other studies. After incubation in 6-OH-DA containing media similar changes were observed in the axonal enlargements, starting already after 30 min of incubation. At this time-point there is a considerable reduction of endogenous NA and a severe damage of the membrane pump uptake mechanism. Incubation with 6-OH-DA and subsequent rinsing for 2 hr caused marked changes, including partly swelling of axons and partly shrinking of the axons into electron dense bodies.The fluorescence histochemical and biochemical results are in good agreement with the ultrastructural studies demonstrating a rapid loss of NA from the adrenergic nerve terminals and main axons and a long lasting depletion of the NA, with a gradual recovery to 75% 6 weeks after the injection.The investigation has been supported by research grants from the Swedish Medical Research Council (14X-2295, 14X-2887 and 04X-3881) Karolinska Institutet, Magnus Bergvalls and Carl-Berthel Nathorst Stiftelser. For generous supplies of drugs we are indebted to the following companies: AB Hässle (6-OH-DA, through Dr H. Corrodi), Pfizer (Niamid^®), Ciba (Serpasil^®). The skilful technical assistance of Miss Bodil Flock, Mrs Waltraut Hiort and Mrs Eva Lindqvist is gratefully acknowledged.