Electron microscopic and morphometric study of the effects of ENKAD preparation on photoreceptor cells of the retina in Campbell rats with hereditary retinal dystrophy

The drug , a complex of ribonucleotides, which was found to improve the vision in some patients with retinitis pigmentosa, stimulates the morphogenesis of rod outer segments (ROS) in mutant Campbell rats with inherent retinal dystrophy. Four litters of neonatal rats, which being subdivided into groups, were injected subcutaneously with either 0.1 ml of 3.5% drug (experimental group) or 0.1 ml of 0.9% NaCl (control group) daily for 6-7 days. The results were checked in posterior area of the retina at Day 7 by the method of electron microscopy and morphometry. The drug treatment resulted in the 12-20 per cent increase of ROS disc number as compared with that in control group. Therefore, photoreceptors are the first retinal cells to be stimulated by the complex of ribonucleotides in rats.     

Sexual difference in LH-cells of the neonatal rats as revealed by immunocytochemistry

Summary Localization and number of pituitary LH-cells were studied in neonatal male and female rats (from the birth to 12th day) applying anti-HCG serum in immunoenzymological procedures. The cells increased in number with developing age after birth. The cells in males and females were equal in number until 4 days of age, whereas thereafter the increase of the cell number in females exceeded that in males. After birth, the cells are mainly concentrated ventrally, being ventro-lateral in the anterior region but converging into the medial-ventral area in the posterior part of the gland. Some dispersion in a dorsal direction is also noted in the latter region. At birth the cells begin to appear in the dorsal area in the anterior portion, as well as in the posterior portion, particularly in the area close to the intermediate lobe and in the zone adjacent to the residual lumen. This was particularly evident in females after 4 days of age. Thus it is concluded that in rats the sexual differences in the pituitary become apparent after the 4th day of postnatal life.     

Development of muscarinic cholinergic receptor binding in the visual system of monocularly deprived and dark reared rats

In 25 day old rats monocularly deprived by unilateral eyelid suture on postnatal day 10 (MD), [³H]quinuclidinyl benzylate (³H-QNB) binding was significantly reduced in the visual cortex (VC) of both sides, but elevated in both superior colliculi (SC). Muscarinic receptor binding in the frontal cortex (FC), a non-visual brain area, in the lateral geniculate nucleus (LGN), and in the retina was not affected. In 25 day old rats raised in complete darkness from birth (DR) similar changes in³H-QNB binding were found in VC and SC. However, binding levels were also decreased in the FC and significantly increased in the retina. In adult (6 month old) MD and DR rats the differences in³H-QNB binding as compared to age-matched controls had disappeared completely in all visual brain areas studied. Detailed Scatchard analyses indicate that the alterations in the³H-QNB binding were due to changes in receptor number only.This paper is dedicated to Dr. Derek Richter on his seventy-fifth birthday.     

Light microscopic radioautographic study on the DNA synthesis of prenatal and postnatal aging mouse retina after labelled thymidine injection.

The DNA synthesis of mouse retina from the 19th prenatal day through 12 months postnatal has been studied by light microscopic radioautography after the injection of tritiated thymidine. A peak of the labeling index after incorporation of tritiated thymidine was found at fetal day 19. The labelled cells decreased gradually with the developing of the eye from the first postnatal day and were completely disappeared in two weeks after birth. The data also indicated obvious regional differences of the incorporation of tritiated thymidine during the periods of the retina development. The labeling index was the greatest in the anterior region compared to the equator region and the posterior region in the same group of age. The average number of the silver grains in labelled nucleus lead to a decrease with the development of the retina after birth, but there was no significant regional differences found in the same group of age. The data shown from this study suggest that the cell differentiation in mouse retina proceed from posterior to anterior region.     

Changes in the properties of photostimulated cyclic nucleotide phosphodiesterase from the retina of rats with hereditary retinal degeneration

A degree of extractability and activation of cGMP-phosphodiesterase (PDE) (EC. 3.1.4.17) from the rod outer segment membranes was studied in Campbell rats with inherited retinal degeneration and control Wistar rats as compared to the control, the PDE extractability in the diseased rats was found to be considerably lower, which manifested as early as the 15th day of the postnatal life. Changes in the GTP-stimulated and basal PDE activity were observed in Campbell rats. Beginning from the 25th day of the postnatal life the GTP-stimulated PDE of degenerative retina decreased and by the 60th day it reached the basal activity level in these animals. In the diseased rats the first 57 days of postnatal life the basal activity of PDE was sufficiently higher, followed by a sharp decrease reaching the basal activity level of the control rats. The obtained data on the changed PDE activity are likely to be a result of the disturbance in the protein-lipid interaction and a change in the external layer of the photoreceptor membranes in rats with inherited retinal degeneration.     

Age-related features of the state of plasma lipoproteins in rats with hereditary retinal degeneration

The content of lipids and lipoproteins was determined in the blood plasma of Campbell rats with inherited retina degeneration and Wistar rats used as control. It was shown that in the 30-day old Campbell rats the content of cholesterol in high density lipoproteins sufficiently exceeded that of control. When the distribution of lipoproteins particles in the density gradient was studied after ultracentrifugation, some "anomalous" particles were revealed in the 30-45 day old rats with inherited retina degeneration, which with respect to the flotation rate occupy intermediate position between the low and high density lipoproteins. The obtained data show that the development of inherited retina degeneration in rats involves a disturbance, of the lipoprotein spectrum of blood plasma.     

Effect of experimental arrest of eye growth on the proliferation and polyploidization of the retinal pigment epithelium in rats

Following the lens removal from the left eye of the newborn rats, animals were obtained with one normal (control) and another microphtalmic eye. The animals were sacrificed on the 2nd, 3rd, 5th, 7th and 9th days of postnatal development after four injections of 3H-thymidine during 19 hrs. The number of labelled nuclei and mono- and binuclear cells in the central zone of the eye fundus was counted on the autographs. After the initial increase of the index of labelled nuclei in the operated eyes (on the 2nd, 3rd and 5th days) it fell below the control level (on the 7th and 9th days). The number of binuclear cells in the operated eyes, as well as in the control, attains on the 5th day 50% of the total number of cells and remains at this level up to the end of the experiment, whereas in the control eyes the number of binuclear cells increases up to 60% on the 7th and 80% on the 9th day. The results obtained have shown that in rats the factors of total eye growth participate in the control of proliferative activity and polyploidization of the pigment epithelium cells in the retina.     

Distribution of transferrin and transferrin receptor of the eype 1 in the process of formation of the rat eye retina in early postnatal ontogenesis

By the method of indirect immunohistochemistry, distribution of transferrin and of transferrin receptor of the type 1 (TFR1) was studied in the formed rat eye retina at the period of early postnatal ontogenesis (from birth to opening of eyelids). It has been established that the character of distribution of these proteins and intensity of specific staining change dependent on the retina formation stage. Retina of the newborn rat is characterized by diffuse transferrin distribution in nuclear retina layer (in the neuroblast layer-NBL) and in the ganglionic cell layer (GCL) as well as in the eye pigment epithelium (PE); relative immunoreactivity to transferrin is not high. At the 5th postnatal day, immunoreactivity to transferrin is maximal and is revealed both in nuclear and in plexiform layers of retina and in the eye PE, the greatest signal being characteristic of NBL. At the 10th postnatal day the transferrin signal intensity in retina decreases, specific staining is revealed in GCL, PE, and in the area of formed outer segments of photoreceptors. At the 15th postnatal day, transferrin is revealed in GCL, in outer and inner photoreceptor segments and in the eye PE. TFR1 is present in all retina layers at all stages of the retina formation; the relative immunoreactivity to TFR1 sharply rises beginning from the 10th postnatal day; correlation between distribution of transferrin and TFR1 is detected in the entire retina of newborn rats as well as in the external retina area at subsequent stages of its development. A possible role of transferrin at various stages of formation of retina is discussed.     

Cyto- and synaptogenesis in the arcuate nucleus of the rat hypothalamus during fetal and early postnatal life

Summary Morphogenesis of the arcuate nucleus of the rat from the 15th fetal day to the 6th postnatal day was investigated light and electron microscopically. The arcuate neurons exhibit a gradual development after the 15th fetal day. All cytoplasmic constituents are present in these nerve cells already during the last days of gestation. Nevertheless, they are not fully differentiated at birth. The first synapse-like structures (presynapses) were observed in 17 day-old, the first synapses in 18 day-old fetuses. During the early postnatal period the number of presynapses decreases, but at the same time there is a gradual increase in the number of the relatively mature synapses. This process starts already during the last days of prenatal life. Although all structural elements of the arcuate nucleus of the adult rat appear to be present at birth, the extent of the neuropil area and the number of the presynapses indicate that the arcuate nucleus is still in a fairly undeveloped stage during the first postnatal days.     

移植视网膜NOS阳性神经元的发育

目的 观察不同年龄组段大鼠正常视网膜及移植视网膜内NOS阳性神经元的发育情况及其定位分布。方法 实验分正常视网膜发育组和移植视网膜发育组,应用还原型烟酰胺腺嘌呤二核苷酸磷酸（NADPH）组织化学方法显示。结果 1、NOS阳性神经元最早出现于生后第五天（P5）,P18时阳性神经元数目达到最高峰,2、移植视网膜具有正常视网膜的各层结构和相似的生长规律,NOS阳性神经元在生后第4天移植视网膜（TP4）中出现,TP12数量达到高峰值,TP22后降至正常成年鼠水平。结论 根据NOS阳性神经元的定位,分布,推测其为无长突细胞,移位无长突细胞及节细胞。     

Temporal and spatial dimensions of postnatal growth of the mouse urinary bladder urothelium

Postnatal growth and renewal of mouse urothelium start on the day of birth. In the present study, temporal and spatial dimensions of urothelial growth were studied during the first two postnatal weeks. Quantitative analysis showed that the rate of urothelial cell proliferation is significantly higher during all 14 postnatal days than in adult mice. Three peaks of proliferative and mitotic activity were revealed: on the day of birth and postnatal day 1, on days 6 and 7, and on day 14. The high proliferation rate around the day of birth and at postnatal days 6 and 7 coincides with cell death in the urothelium. Semiquantitative analysis showed that during all 14 postnatal days, the urothelial proliferative response is mostly confined to the basal cell layer. Urothelial cells divide predominantly in parallel to the plain of the urothelium on all chosen postnatal days. Increased portions of urothelial cells, dividing perpendicularly to the urothelium were observed only on the day of birth and on postnatal day 7. Our results suggest that postnatal growth of mouse urothelium is particularly the result of an increasing number of cells in individual cell layers and not the result of an increasing number of cell layers.     

Evolution of the retinal distribution of amacrine cells immunoreactive to phenylethanolamine-N-methyltransferase during postnatal development in the white rat

The phenylethanolamine-N-methyltransferase immunoreactive cells were demonstrated by the first postnatal day in whole-mounted retinas. They were distributed all over the retina in young rats (more numerous in the superior temporal quadrant) and were restricted to the superior retina in adult rats.     

Study on RNA synthesis in the retina and retinal pigment epithelium of mice by light microscopic radioautography.

With the aim of determining the distribution of the incorporation of 3H-uridine in both retina and retinal pigment epithelium (RPE), the mouse eyes at embryonic day 9.5 (E 9.5), E 12.5, E 14.5, E 16.5, E 18.5 of gestational ages, and postnatal day 1 (P 1), P 3, P 7, P 14 were analyzed by light microscopic radioautography. Small pieces of the ocular tissues were labelled with 3H-uridine in vitro and light microscopic radioautographs were prepared. The average grain numbers per cell of the respective regions of tissues were calculated. In the retina, the grain numbers increased gradually from E 9.5 to P 1 and reached the maximal value at P 1, and then decreased until P 14. However, the grain numbers were more in the vitreal portion than those in the scleral portion at E 16.5 and then became more in the scleral portion from E 18.5 to P 14. It is considered that the ganglion and bipolar cells finish the RNA synthesis earlier, while the photoreceptor cells do it later during the fetal and postnatal development. In the RPE, the grain numbers gradually increased from E 12.5 to P 7 and then decreased until P 14. Considering the same ages, the grain numbers increased in the following order, anterior, equatorial and posterior regions during embryonic stages, but decreased in the same order after birth. Therefore, it is suggested that the activity of RNA synthesis in PE cells is higher in the posterior region than in the anterior region during embryonic stages. But the activity ascends generally and becomes relatively higher in the anterior region, after birth. Comparing the retina and RPE, it was noted that the grain numbers in the RPE were more important than in the retina and that the maximal value was at P 1 in the retina, while it was at P 7 in the RPE. From these results, it can be concluded that the RNA synthesis ceases earlier in the retina than in the RPE.     

Ontogeny of histamine-immunoreactive cells in rat stomach

Summary An antiserum against hemocyanin-conjugated histamine was used to study the cellular stores of histamine in the stomach, especially the oxyntic mucosa, of fetal and early postnatal rats. Tissues were fixed in 4% 1-ethyl-3(3-dimethyl-aminopropyl) carbodiimide (EDC-DI) and standard immunofluorescence technique was used. Histamine was first detected on the 16th embryonic (E16) day when a few histamine-immunoreactive (HA-ir) cells and nerve fibers were observed in the muscular layer of the stomach wall. On day E18, HA-ir cells were visualized for the first time in the oxyntic mucosa of the stomach, and from that day on the number of such cells increased slowly initially and after day E20 more rapidly. At birth many of the HA-ir cells in the oxyntic mucosa possessed processes giving them a paracrine-like appearance typical of enterochromaffin-like cells (ECL cells). Only a very small number of the HA-ir cells represented metachromatically stained mast cells and were located in the submucosa. After birth, the number of HA-ir ECL cells increased steadily, until day 21 when the distribution and number was very similar to that of the adult. The results suggest that histamine-containing neurons and ECL cells appear in the stomach wall before birth, and that there are histamine-containing ECL cells in the mucosa and mast cells in the submucosa of the stomach wall at birth.     

Post-natal maturation of the retina in the albino rat. I. The pigment epithelium

The Authors studied the postnatal development of the retinal pigment epithelium in the albino rat, in order to elucidate its morphological and functional evolution, correlated to the numerous functional roles played in Vertebrates (Scheme 1). At birth, epithelial cells show few cytoplasmic organules and the apical surface provided of small depressions. From the third to the fifth postnatal day the first apical microfolds surround the depressions. From the seventh to the ninth day inner segments develop, whilst the apical surface of the epithelial cells is covered by many finger-like microfolds. During the eleventh postnatal day the buds of the outer segments and many lamellar microfolds can be demonstrated. During the sixteenth day the retina reaches its adult morphology. It is therefore well-evident that birth, similarly to many other Vertebrates, is not the last step, but only a moment, in the development of the retina: this process is completed only during postnatal life, when environmental light is able to stimulate every ocular structure.     

NADPH-Diaphorase Activity in Normally Developing and Intracranially Transplanted Retinas

The activity and distribution of nicotinamide dinucleotide phosphate diaphorase (NADPH-d), an enzyme that is widely distributed in the central nervous system and involved in the production of the free radical nitric oxide, were investigated histochemically in the normal developing and intracranially transplanted retinas. In the normal rat retina, NADPH-d activity was first detected in cells in the ganglion cells layer (GCL) and blood vessels on the first postnatal day (P0). A small but distinct population of NADPH-d positive cells were observed along the inner border of the inner nuclear layer at P7. NADPH-d positive sublaminae began to appear in the inner plexiform layer during the second postnatal week, and several strongly reactive sublaminae resembling those observed in the adult were observed by the fourth postnatal week. The overall spatio- temporal sequence of development of NADPH-d positive cells in the transplanted retina was similar to that of the normal retina, except a lack of reactive in the inner plexiform layer in more mature transplants as compared with normal retinas of corresponding ages. These results indicate that the time course of development and distribution of NADPH-d cells in early postnatal retina requires signals mainly of intraretinal origin and is independent of influence from the surroundings. While this finding is supportive to the notion that neurons that are rich in NADPH-d are resistant to injury or perturbation, the observation of a lack of well organized NADPH-d reactive sublaminae in the inner plexiform layer in older transplants suggests a possible alteration in the synaptic circuitry in the inner retina with increasing postgrafting survival time.     

Decrease of opsin content in the developing rat photoreceptor cells by systemic administration of L-glutamate.

L-Glutamate, a putative photoreceptor cell neurotransmitter, causes thinning of the inner layers of the retina and has been used for preparing biologically fractionated photoreceptor cells. However, it is possible that absence of the inner retinal layers may affect the remaining retina, and/or glutamate may directly affect photoreceptor cells. We evaluated quantitatively the effects of L-glutamate on the developing photoreceptor cells by measuring the rod photoreceptor cell-specific protein, opsin. We purified rat rhodopsin and used it as the standard for measuring opsin content of rat retinas with competitive enzyme-linked immunosorbent assay. Various concentrations of glutamate were injected into 7-day-old rats, and the effects of the amino acid concentration on opsin expression were determined on postnatal day 14. Inner layers of the retina degenerated when 10 microliters or 15 microliters of 2.4 M glutamate/gram body weight was administered subcutaneously. Opsin content of these glutamate-treated retinas decreased significantly compared with control retinas. We administered glutamate to rats at various stages of development and determined the effects by light microscopy on postnatal day 14. The administration of glutamate resulted in no degeneration of the inner retina if injected on postnatal day 1 or 2, degeneration of the inner retina between day 3 to 7, and again, no degeneration after postnatal day 13. Opsin content decreased significantly when glutamate was administered between postnatal day 1 to 7, but not after day 13, the day the blood-retinal barrier seems to reach maturity. Our findings indicate that systemic administration of L-glutamate affects the expression of opsin in the developing rod photoreceptor cells.     

Development of substance P and neurokinin A immunoreactivity in ganglia supplying nerves to the submandibular glands of the rat

Developing submandibular, trigeminal and superior cervical ganglia, which provide innervation to the submandibular glands, were studied for substance P (SP)-and neurokinin A (NKA)-immunoreactive (IR) ganglion cells and nerve fibres in rat. These ganglia were examined by using an indirect immunofluorescence technique at daily intervals from the 16th day in utero (i.u.) until birth, and subsequently on the 2nd, 5th, 7th, 12th, 16th, 30th, 42nd postnatal day and in the adult (3 months). In the submandibular ganglion SP- and NKA-IR cells and fibres first appeared in considerable numbers on the 19th day i.u. (in one sample out of five on the 18th day i.u.), when more than 90% of the ganglion cells were immunoreactive to SP and NKA. The number stayed at more than 90% to the 7th postnatal day and then slowly decreased to the levels of adult animals (18% SP, 17% NKA). The first SP- and NKA-IR ganglion cells and fibres appeared in the trigeminal ganglion on the 18th day i.u. when they represented 7% (SP) and 4% (NKA) of the ganglion cells. The number of SP- and NKA-IR cells increased steadily, reaching a maximum at the time of birth when 68% (SP) and 74% (NKA) of the ganglion cells were immunoreactive. Thereafter they began to decrease toward the level of an adult rat (10% SP, 11% NKA). In the superior cervical ganglion only a few SP-and NKA-IR ganglion cells were detected from the 19th day i.u. to the fifth postnatal day. Positive ganglion cells were also occasionally found in the nerve trunks outside the superior cervical ganglion. From the seventh day onwards no SP- or NKA-IR ganglion cells were found. SP-and NKA-IR SIF (small intensively fluorescent) cells were detected from the 16th postnatal day onwards.     

Delayed maturation of the male cerebral cortex in rats.

45 male and female Wistar rats were given a single injection of 3H-thymidine (10 mu Ci/g body weight) on day 1, 7, 14 or 21. All animals survived until 60 days of age when they were perfused with 10% neutral formalin and the brains were removed and prepared for autoradiography. The sagittal section of the cortex (L980 micron) was 6.8% larger in the males (p less than 0.05) but the packing density of the cortical cells was 5.9% higher in the females (p less than 0.01), thus bringing the total number of cells to the male levels. The diameter of the female cortical cells was 3.8% smaller than those of the males (p less than 0.05). The greatest difference was among the smaller cells (3-9 micron). The rate of postnatal acquisition of cortical cells was indicated by the number of radioactive-labelled cells. Males had more labeled cells after each injection; it was most pronounced (32% difference) on day 7 (p less than 0.05). This may reflect a delayed acquisition rate of cells formed before birth, since more cells could be labeled by the postnatal injection.     

Proliferative activity of cells of the intermediate lobe of the rat pituitary during the postnatal period.

Cellular proliferation was studied in the intermediate lobe (IL) of the pituitary gland of developing rats by labelling cells at the S-phase of the cell cycle with bromodeoxyuridine (BrdU). The number of BrdU-labelled cells in the IL decreased from birth until the 14th postnatal day and was low from that day until the end of the first month after birth. Throughout the postnatal period a large proportion of BrdU-labelled cells was found in the marginal layer (ML) of the IL, suggesting for the ML a role as a germinative layer of the IL during postnatal growth. Double immunostaining with anti-BrdU and anti-MSH showed that MSH cells actively proliferate as from the day of birth. Cells doubly immunostained with anti-BrdU and anti-S100 protein were first seen on the 14th postnatal day. From then onwards, most proliferating cells were labelled with either anti MSH or anti S-100 protein. This, together with the high proportion of proliferating cells found in the ML marks a clear difference with the pattern of cellular proliferation previously reported during a similar period in the anterior lobe of the rat pituitary.