Weeks In Oculo Research Articles

The oestrogenized rat myometrium inhibits organotypic sympathetic reinnervation

Chronic administration of oestrogen to rats during the infantile/prepubertal period provokes, at 28 days of age, complete loss of noradrenaline-labelled intrauterine sympathetic nerves. It is not known whether oestrogen inhibits the growth or causes the degeneration of developing uterine sympathetic nerves, or whether the uterus recovers its innervation following cessation of infantile/prepubertal oestrogen treatment. In the present study, we analysed the time-course of the effects of oestrogen on the development of uterine sympathetic nerves in the rat, using histochemical methods. In addition, the pattern of sympathetic reinnervation of the uterus of intact and ovariectomised females was assessed 3 and 6 months after cessation of chronic oestrogen treatment. The ability of sympathetic nerves to reinnervate the oestrogenized uterine tissue was assessed in intraocular transplants of uterine myometrium into ovariectomised host rats. Early exposure to oestrogen did not inhibit the approach of sympathetic nerves to the uterus, but prevented the normal growth and maturation of intrauterine sympathetic fibres and abolished the innervation that reached the organ before initiation of treatment. Three or six months following cessation of oestrogen treatment, most of the sympathetic nerves were restricted to the mesometrium and mesometrial entrance, whereas intrauterine innervation remained persistently depressed as a consequence of a sustained oestrous-like state provoked by ovarian dysfunction (polycystic ovary). An organotypic regrowth of uterine sympathetic nerves was observed in ovariectomised infantile/prepubertal oestrogen-treated animals. After 5 weeks in oculo, the innervation of oestrogenized myometrial transplants was reduced by 50%, and substantial changes in the pattern of reinnervation were observed. In control transplants, 86% of the nerves were terminal varicose myometrial and perivascular nerve fibres, whereas 14% were preterminal nerve bundles. In oestrogenized myometrial transplants, 83% of the noradrenaline-labelled intercepting nerves were enlarged preterminal bundles and only 17% were terminal fibres. These results indicate that the oestrogenized myometrium is unattractive for sympathetic nerves and inhibits organotypic sympathetic reinnervation.

Binding of insulin-like growth factors (IGF-I and IGF-II) to the IGF-II/mannose 6-phosphate receptor in fetal rat myocardium.

Previous studies indicate that fetal rat heart tissue contains large amounts of insulin-like growth factor (IGF)-II/mannose 6-phosphate (Man 6-P) receptor messenger RNA, with receptor messenger RNA levels falling by 20 days after birth. We examined the amount of IGF receptor protein in developing rat myocardium. To establish a model in which the role of neural, hormonal, and hemodynamic controls of IGF receptor binding could be studied, we compared binding of IGF-I and IGF-II in normally growing rat atria and ventricles with embryonic day 12 (E-12) atria and ventricles maturing in the anterior eye chamber of an adult host rat. In oculo, embryonic myocardium matures without hemodynamic load or exposure to the fetal hormonal milieu. In fetal rat hearts (E-12 to E-19), both IGF-I and IGF-II intensely bound to a protein with a molecular weight corresponding to the IGF-II/Man 6-P receptor. Receptors were identified using sodium dodecyl sulfate/polyacrylamide gel electrophoresis autoradiography and western blot analysis using Ab3637, a specific polyclonal antibody against rat IGF-II/Man 6-P receptor antigens. This antibody competed for binding of both IGF-I and IGF-II to the band with molecular radius corresponding to 260,000 (reduced). In normally growing rat atria, IGF-I binding to the IGF-II/Man 6-P receptor was similar to ventricular tissue; however, there was significantly greater binding of IGF-II than of IGF-I in both atrial and ventricular tissue. High levels of IGF-II binding to the IGF-II/Man 6-P receptor were observed in both fetal rat atrial and ventricular grafts until 6-8 weeks in oculo. As in normally growing heart tissue, there was similar IGF-I binding to the IGF-II/Man 6-P receptor in atrial grafts compared with ventricular grafts from 2-8 weeks after implantation. For the first 2 weeks after grafting, the ventricular grafts had relatively higher IGF-I binding to the IGF-II/Man 6-P receptor compared with later time points examined. The present data indicate that atrial and ventricular binding of IGFs to the IGF-II/Man 6-P receptor decreases with age, suggesting that decreased IGF binding may be independent of postnatal hemodynamic changes. The decrease is similar in in oculo embryonic rat cardiac grafts and normally growing heart tissue.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of sympathetic innervation on size of myocytes in embryonic rat heart cultured in oculo

Embryonic rat myocardium cultured in the anterior eye chamber of an adult rat increases in mass and differentiates into mature myocardium by most morphological criteria [ 1]. When sympathetic innervation of grafted heart tissue was prevented by superior cervical ganglionectomy (SCGx), growth of the grafts was severely compromised. The present study used morphometric methods to examine the contribution of myocyte size to the differential growth of grafts in sympathetically denervated and intact eye chambers. For this purpose, atria or ventricles from 12-day gestation rat hearts were grafted into sympathetically denervated and intact eye chambers of male host rats. Tissue was harvested after either 2 or 8 weeks in oculo. Myocyte diameter and the ratio of cytoplasmic area to nuclear area increased between 2 and 8 weeks in oculo in both atrial and ventricular grafts. In these unloaded heart grafts, estimated myocyte size did not differ between atrial and ventricular myocytes. Grafts into sympathetically innervated and denervated eye chambers did not differ in myocyte diameter or in the ratio of cytoplasmic to nuclear area, suggesting that myocyte size cannot explain the smaller mass of grafts in sympathetically denervated eye chambers. Thus, it is likely that other factors such as myocyte proliferation, myocyte survival or altered tissue composition underlie the lesser growth of grafts not innervated by sympathetic nerves.

Intraocular hypothalamic transplants containing histaminergic neurons: Innervation of host iris and hippocampal cografts

Hypothalamic tissue containing the tuberomammillary nucleus was dissected from fetuses of Embryonic Day 17 and inserted into the anterior chamber of the eye of young adult recipient rats. The growth of hypothalamic grafts was monitored through the translucent cornea and transplants were found to double in size over the 8 first weeks in oculo. After 4 weeks fetal hipocampal formation (Embryonic Day 18) was inserted into the eye chamber in half of the previously grafted animals and placed in contact with the first grafts. Double grafts were allowed to mature for up to 18 weeks before sacrifice. Recipient rats were anesthetized and superfused with carbodiimide and paraformaldehyde, after which transplants were removed, frozen, sectioned on a cryostat, and incubated with histamine antibodies. Immunohistochemical evaluations revealed a large number of histamine-positive nerve cell bodies with processes innervating the entire hypothalamic graft with a dense plexus of varicose fibers. Such histamine-positive fibers were also seen to invade the surrounding host iris in some cases with thick axon bundles as well as with single fibers. When hypothalamic transplants were combined with hippocampal grafts numerous histamine-immunoreactive fibers invaded the hippocampal tissue to form a plexus of varicose terminals throughout the cografts. After 4 weeks in oculo only a sparse histamine-positive innervation of hippocampal grafts was found, while 18-week-old double grafts contained a considerably larger amount of immunoreactive neurites. Thus, it appears from preliminary studies that the histamine-containing neurites continually innervate hippocampal grafts in a pattern described for other monoamine neurons as well. The present findings suggest that histaminergic neurons survive transplantation to the anterior chamber of the eye and can provide both host iris and cografted hippocampal formation with a dense plexus of histamine-containing fibers.

Genetic, neurohumoral, and hemodynamic influences on spontaneously hypertensive rat heart development in oculo.

To distinguish among genetic, neurohumoral, and hemodynamic explanations for structural and functional differences in the hearts of young spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) control rats, embryonic SHR and WKY rat heart tissue was cultured in the anterior eye chamber of adult SHR and WKY rats. In study 1, atria from E-12 WKY rat embryos grafted into anterior eye chambers of either SHR or WKY host rats achieved a larger size than did SHR grafts by 8 weeks in oculo (2.98 +/- 0.75 and 2.55 +/- 0.32 mm2 vs. 1.80 +/- 0.20 and 2.04 +/- 0.44 mm2). Beating rates did not differ between SHR and WKY rat atria implanted into SHR or WKY host rats. In study 2, ventricles from E-13 embryonic SHR and WKY rat hearts grew to similar size and weight when implanted into SHR or WKY host rats (e.g., SHR hearts, 1.81 +/- 0.32 vs. 1.74 +/- 0.33 mm2; WKY rat hearts, 1.75 +/- 0.29 vs. 2.29 +/- 0.32 mm2). Ventricle grafts from SHR embryos into SHR host rats beat more rapidly (165 +/- 19 beats/min) during weekly measurements than either WKY rat ventricles (92 +/- 9 beats/min in SHR hosts and 99 +/- 9 beats/min in WKY host rats) or SHR ventricles grafted into WKY host rats (109 +/- 7 beats/min, p less than 0.001). In study 3, atria from E-13 SHR and WKY rat embryos were grafted into sympathectomized and intact eye chambers of SHR or WKY host rats. Sympathectomy of the eye chamber compromised growth of grafts into WKY host rats (1.54 +/- 0.24 vs. 0.90 +/- 0.14 mm2) but not SHR hosts (1.54 +/- 0.25 vs. 1.73 +/- 0.24 mm2). Grafts into sympathectomized eye chambers of WKY host rats beat more slowly than grafts into eye chambers with sympathetic innervation intact (282 +/- 14 vs. 202 +/- 14 beats/min); sympathectomy did not alter beating rate of grafts in SHR hosts (266 +/- 14 vs. 255 +/- 18 beats/min). These results suggest that the growth and beating rate of SHR atrial grafts may be less sensitive to sympathetic innervation than WKY rat atrial grafts. In these studies, SHR grafts did not grow larger than WKY heart grafts and did not show an increased intrinsic beating rate, suggesting that the cardiac hypertrophy and increased intrinsic beating rate observed in intact SHR are unlikely to result from direct genetic programming.

Morphological development of the rat heart growing in oculo in the absence of hemodynamic work load.

We evaluated cardiac muscle development in the absence of hemodynamic work load but in the presence of host factors including blood vessels, nerves, and circulating neurohumoral agents by transplanting 12-day fetal rat ventricle into the anterior eye chamber of adult host rats. Implants were studied by electron microscopy at intervals from 1 to 14 weeks in oculo. For comparison with myocardium developing in oculo, 12-day fetal tissue and 3-, 8-, and 28-day-old normally growing rats were also studied. At 1 week in oculo, myofibrils were laterally located and more frequent than in the 12-day fetus. Fibrils had clear Z bands and H bands, but no M bands. At 10 days in oculo (comparable to birth in normally growing animals), myocyte mitoses were present and tritiated thymidine autoradiography revealed many labeled myocyte nuclei. By 5 weeks in oculo, cells were filled with mature myofibrils with clear M bands and lateral connections between adjacent Z bands. However, myofibril bundles sometimes coursed at sharp angles to each other within single cells. Except for the relative lack of fibrillar polarization and small cell size, ultrastructure of myocytes developing in oculo for 5 or more weeks appeared very similar to myocytes developing in normally growing rats. By 10 weeks in oculo, when in situ growing hearts are clearly in a hypertrophic phase of growth, no mitoses or tritiated thymidine-labeled nuclei were present in myocytes, although labeled nonmyocyte nuclei were present. Morphometric evaluation revealed no change in myocyte diameter or nuclear-to-cytoplasmic ratio from 1 to 3 weeks in oculo, consistent with continued hyperplastic growth. Binucleated cells were present by 3 weeks in oculo and later, and the cytoplasm per nucleus increased fourfold between 3 and 5 weeks in oculo, suggesting conversion to hypertrophic cell growth. We concluded that cells proliferated and differentiated in the absence of a hemodynamic load, but that polarized alignment of myocytes and myofibrils was incomplete.

Sympathetic innervation alters growth and intrinsic heart rate of fetal rat atria maturing in oculo.

The influence of sympathetic innervation on the growth and intrinsic rate of beating established by fetal rat heart was studied by culturing fetal atrial tissue in sympathetically innervated and denervated anterior eye chambers of adult Sprague-Dawley rats. One anterior eye chamber in each host rat was sympathetically denervated by removing the ipsilateral superior cervical ganglion. In oculo, atrial grafts were vascularized by blood vessels sprouting from the iris and innervated by sympathetic and parasympathetic fibers from the ground plexus of the iris. Innervation was assessed by light-activated efferent nerve stimulation to the grafts that changed their rates of beating. The norepinephrine contents of 16 atria cultured for 2.5 months in sympathetically innervated and denervated eye chambers were 5.7 +/- 1.1 ng/implant vs. 0.2 +/- 0.07 ng/implant (mean +/- SEM), indicating permanent sympathetic denervation of the anterior eye chamber and the implanted atria. By 8 weeks in oculo, atria maturing in sympathetically innervated anterior eye chambers were 86% larger than those in denervated eye chambers (2.22 +/- 0.29 vs. 1.19 +/- 0.13 mm2); the weight of innervated transplants was over 3 times that of noninnervated grafts (2.35 +/- 0.75 vs. 0.76 +/- 0.21 mg). After implanted atria had ceased growing rapidly (2.5 months in oculo), bipolar electrodes were implanted adjacent to the cornea to record impulses from atrial grafts while host rats were unanesthetized. The dark-adapted baseline heart rates of sympathetically innervated and noninnervated atria were virtually identical (289 vs. 290 bpm). Graft intrinsic heart rate was estimated by combined beta-adrenergic and muscarinic receptor blockade with atenolol (1.0 mg/kg) and methylatropine (10 micrograms/kg). Sympathetically innervated transplants had lower intrinsic heart rates than noninnervated atria (134 +/- 25 vs. 213 +/- 12 bpm). These data suggest that sympathetic innervation of the developing heart influences both growth and intrinsic rate of beating.

Release of serotonin from human carcinoid tumor cells in vitro and grown in the anterior eye chamber of the rat.

Serotonin (5-HT)-producing human carcinoid tumors have been successfully transplanted to the anterior eye chamber of cyclosporine treated rats. Tumor transplants were rapidly vascularized and viable tumor cells could be demonstrated in transplants grown for 2 to 3 weeks in oculo. The release of 5-HT from carcinoid tumor cells grown in the anterior eye chamber and from tumor cells in suspension was studied after stimulation with various adrenoceptor agonists. A dose dependent release of 5-HT upon stimulation with isoprenaline was demonstrated for all viable carcinoid tumors in oculo. The isoprenaline-stimulated release of 5-HT from the tumor cells was further studied in vitro, and was efficiently antagonized by propranolol or nadolol, but not by phenoxybenzamine, indicating activation of beta-adrenoceptors on carcinoid tumor cells upon isoprenaline stimulation. Incubation of tumor cells with verapamil, a calcium channel inhibitor, also prevented the release of 5-HT upon isoprenaline stimulation. Thus, carcinoid tumors can be successfully transplanted to the anterior eye chamber of cyclosporine treated rats, offering new possibilities to study the pharmacologic and biologic features of this tumor. In the future, carcinoid tumors, as well as other endocrine tumors, may be grown in oculo in rats and receptor pharmacology and individual sensitivity to cytotoxic drugs can be studied, thus enabling optimal clinical treatment.

Immature and mature neurofilament-immunoreactive trigeminal fibers can innervate the iris as studied by intraocular grafting of iris and trigeminal ganglia

Using immunohistochemistry on stretch-prepared whole mounts of adult rat irides, a dense, well-organized plexus of neurofilament-positive nerves originating in the trigeminal ganglion can be visualized. Such a two-dimensional tissue preparation is well-suited for studies on sensory and autonomie nerve fiber growth. In the present study the growth capacity of such neurofilament-positive nerves has been studied immunohistochemically. In irides homologously transplanted to the anterior eye chamber of adult albino rats, the intrinsic neurofilament-positive network had almost completely disappeared 4 days postoperatively. In whole mounts of iris grafts after 15 days and 4 weeks in oculo a gradually increasing plexus of nerves was observed. After 3.5 months in oculo a dense, regular network of fluorescent fibers had formed in the iris grafts to the same magnitude as in situ. However, whereas large axon bundles constituted a prominent feature of the distribution of neurofilament-positive nerves in situ, only a few and relatively thin axon bundles were seen in the grafts. The growth capacity of the neurofilament-positive trigeminal nerves was also studied by grafting fetal trigeminal ganglia to the anterior eye chamber. As visualized in cryostat sections, trigeminal grafts contained a large number of strongly fluorescent perikarya and a high density of positive fibers after intraocular maturation. Such grafts readily innervated the host iris. In the area immediately adjacent to the grafts, thin, parallel, rather weakly fluorescent fibers radiated out from the ganglia. When mature trigeminal grafts with attached host iris were regrafted to the anterior eye chamber of adult animals for a few days, in order to remove the intrinsic host iris innervation, such irides showed outgrowing fibers, often organized in small axon bundles, at long distances from the ganglion graft. The present report shows that both mature and immature neurofilament-immunoreactive neurons are capable of innervating the iris. Furthermore, this ingrowth can occur both in the presence and absence of normal intrinsic neurofilament-positive nerve fibers.

Embryologic development of rat adrenal medulla in transplants to the anterior chamber of the eye

The morphological development and plasticity of embryonic and postnatal rat adrenal medullary cells were studied in homologous adrenal grafts to the anterior chamber of the eye. The eyes of recipient rats were adrenergically denervated 10 days prior to grafting by extirpation of the superior cervical ganglion in order to increase levels of NGF and NGF-like activities in the iris. Grafts taken at the 15th day of embryonic development (E15), i.e., at the beginning of immigration of medullary progenitor cells into the adrenal cortical anlagen, contained no cortical or mature medullary cells after 2 weeks in oculo. Numerous sympathoblastic cells, however, were located at the anterior surface of the iris. E 16 and E 17 transplants showed abundant mature cortical tissue after 2 weeks. Small groups of medullary cells with the ultrastructural characteristics of mature pheochromoblasts or young chromaffin cells were interspersed among cortical cells without forming a discrete medulla. Neuronal cells were exclusively found outside the cortical cell mass. Sympathoblasts grew at the surface of the iris, while young sympathetic nerve cells, which were invested by Schwann cells and received synaptic axon terminals, were embedded into the stroma of the iris. Grafting of E 21 adrenals yielded very similar results except that, in a few instances, young chromaffin cells were located outside the cortex and sympathetic nerve cells were seen to be in close contact with cortical cells. In transplants of adult medullary cells typical mature adrenaline and noradrenaline cells were clearly distinguishable after 8 weeks even in the absence of cortical cells. The only indication of phenotypical changes in these cells was the formation by some of them, of neuritic processes which coul be visualized in glyoxylic acid-treated whole mounts of irises. These results are compatible with the idea that embryonic adrenal medullary cells have the environmentally controlled potential to develop along the neuronal or endocrine line, but could also be interpreted in terms of a selection of a specific subpopulation with predetermined potentialities by a specific microenvironment. Moreover, these results suggest that increasing differentiation of medullary cells is accompanied by progressive restrictions in their genetic program, which eventually prevent full transdifferentiation of mature chromaffin into neuronal cells.

Delayed development of GFA immunoreactivity in intraocular cortex cerebri grafts during thyroid hormone deficiency

Dependency of astrocytes on thyroid hormones during development was studied in intraocular cortex cerebri grafts, using antibodies to the glial fibrillary acidic protein (GFA). Fetal cortex cerebri was left in the anterior eye chamber of normal and thyroidectomized adult recipient rats 3 and 8 weeks to mature. All cortex grafts survived and became vascularized in the eye. The grafts were cryostat-sectioned, and reacted with an antiserum specific for GFA. The density of GFA immunoreactivity in the cortex graft neuropil was subjectively estimated with fluorescence microscopy. After 3 weeks in oculo, the number of positive structures was significantly lower in the thyroidectomized group as compared to the euthyroid control group. This effect of thyroidectomy on the cortex grafts was entirely counteracted by daily injections of thyroxine (100 μg/kg s.c.) to similarly thyroidectomized recipients. The distribution of GFA-positive structures was even within individual grafts of all three groups. Neither were any differences in morphology of fluorescent astroglial processes observed. The overall density of fluorescent GFA-positive structures was considerably higher than that found in age-matched normal in situ counterparts, even in the thyroidectomized group. After 8 weeks in oculo, the difference between the control and thyroidectomized groups in density of GFA-positive structures in the neuropil had disappeared, and no general increase in density of immunoreactivity was noted in the control group. These results indicate that thyroid hormones might normally play a role for the development of GFA in the early postnatal brain, as thyroid hormone deficiency causes a transient delay in development of GFA immunoreactivity in intraocular cortex cerebri grafts.

Distribution of Thy-1-like immunoreactivity in normal, sympathetically denervated and grafted mouse irides.

The distribution of Thy-1-like immunoreactivity was studied in whole-mounts of adult mouse iris and intraocular iris grafts 4 days and 4 weeks postoperatively. After fixation in picric acid/paraformaldehyde, the irides were incubated with a polyclonal rabbit anti-mouse brain Thy-1 antibody. In the adult mouse iris, a dense network of thin bundles and individual fibres was seen on the dilator plate and in the sphincter. Fluorescence paucities, resembling Schwann cell nuclei, were frequently seen along the bundles. Numerous mast cells, stained specifically with the Thy-1 antibody, were scattered over the entire surface of the iris. The ciliary body contained several brightly fluorescent bundles, and some circularly running individual fibres. In 4-day iris grafts, the Thy-1-like immunoreactivity had disappeared, except in mast cells. After 4 weeks in oculo, a regular plexus of thin fibres had reappeared in the iris grafts. Sympathetic denervation of adult irides did not seem to affect the Thy-1 immunoreactivity in terms of either fluorescence intensity or fibre distribution. The present data suggest a distribution of the glycoprotein Thy-1 along nerve fibres in the iris.

Electrophysiological and cytological studies of brain homografts in the anterior chamber of the eye: Maturation of cerebellar cortex in oculo

The intraocular transplantation technique was used to study maturation of cerebellar homografts with electrophysiological and cytological methods. Taking of the transplants was optimal using donors with a gestational age of 15–17 days, whereafter it declined markedly. The time-table for the cytological maturation mimicked the in situ maturation surprisingly well, being retarded at the most by 5 days. External granule cells appeared 5 days postoperatively, and had completely disappeared about 5 weeks later. Internal granule cells appeared from about day 12. Purkinje cells were identified in clusters from day 8. They slowly fanned out to form a monolayer after about 3 weeks in oculo, situated between an internal granule cell layer and a molecular layer. The molecular layer was clearly recognized from the third week and often contained apical dendrites from the Purkinje cells. The overall cytological organization of the mature transplants was very similar to the normal cerebellum, also including some foliation and always including typical trialaminar areas. The intraocular transplantation technique is thus very suitable for producing cytologically homotypical cerebellar transplants. The electrophysiological maturation of the transplants was also followed using extracellular recordings. A spontaneous discharge pattern strongly suggesting the presence of Purkinje cells mimicked the well known in situ activity both regarding time course and way of firing. Thus, few single spikes were recorded 12 days postoperatively having very few high frequency bursts. The interspike intervals and the inter- and intraburst intervals then gradually decreased to reach values very close to those of normal adult cerebellum about 4 weeks postoperatively. Interspike and interburst interval histograms are presented to show these similarities. Also, potentials evoked by stimulating the base or the surface of the transplants showed action potential responses similar to those of normal cerebellum. Thus, (1) stimulating the base of the transplant, a short latency all-or-none single spike was elicited. (2) When stimulating the surface of the transplant often 1–3 driven spikes over a 5–10 msec period could be elicited. This response was invariably graded. (3) Stimulation of the surface often produced a short latency inhibition of spontaneous discharge lasting 40–120 msec. This response was also graded. The inhibition was shown to be independent of adrenergic fiber influence as shown by superior cervical ganglionectomy or parenteral reserpine administration. The inhibition was antagonized by the GABA-blocker bicuculline when administered parenterally. These 3 responses closely resemble the antidromic spike response, the parallel fiber excitation response and the basket-stellate cell inhibition response respectively, as seen in the normal adult cerebellum in situ. It is concluded that the intraocular cerebellar homografts develop and mature in a very homotypical way as shown by means of electrophysiology and histology in spite of the complete lack of normal connections with the rest of the brain and underlines the importance of intrinsic regulatory mechanisms in CNS ontogeny.