RNA and DNA in developing retinae: comparison of a normal with the degenerating retinae of C3H mice

Abstract— The Nucleic acids were measured in developing retinae of normal (DBA) mice and those afflicted with an inherited degenerative disease (C3H). The content of RNA in normal and C3H retinae increased to a maximum at 5 days of postnatal age. Thereafter, that of C3H retinae declined to a value lower than the normal. The content of DNA in normal and C3H retinae was maximal at 10 and 5 days of postnatal age, respectively. By 20 days, it declined in both retinae to nearly adult values. The DNA/RNA ratio of normal adult retinae was about 3, while that of C3H adult retinae was nearly 1–5. It is proposed that the photoreceptor cells possess a smaller cytoplasmic volume and a larger DNA/RNA ratio than the cells of the inner retina. The loss of DNA in developing normal and C3H retinae appears to result from cellular death. It was calculated that approximately 1 million cells in normal and 6 million cells in C3H retinae disappear during development. Cellular death in C3H retinae may not be restricted to the photoreceptor population.     

Changes in glucose and energy metabolism in Vivo in developing retinae from visually-competent (DBA-1J) and mutant (C3H-HeJ) mice

Abstract— The distribution in vivo of glucose and lactate between the complete or sub- divided retina and the blood has been evaluated in DBA and C3H mice during postnatal development. Levels in vivo of several intermediates of glucose and energy metabolism were measured by enzyme-linked fluorometric assays of freeze-dried retinae; glucose and lactate were determined in freshly-drawn plasma. DBA retinae. During the first 20 days of postnatal life, the level of glucose in the plasma rose slightly while that in the retina declined: during this period the level of lactate in the plasma rose and became nearly equal to that in the retina. Changes during development in levels of glucose and glycogen were consistent with the interpretation that the rate of utilization of glucose in vivo is enhanced during early postnatal life. C3H retinae. The levels of glucose and glycogen in vivo were abnormally high throughout the developmental period, whereas levels of lactate were normal. The rise in levels of glucose after the 15th postnatal day was not related to an increase in blood levels of glucose but rather to a decreased utilization of glucose during this period. For the first 10 postnatal days the content of glucose, lactate, ATP and P-creatine within the photoreceptor layer of C3H retinae were within normal limits. Then, biochemical changes occurred which were secondary to ultrastructural pathology in the photoreceptors. This observation suggested that glucose metabolism and energy production are not involved in the primary aetiology of the inherited disease.     

CYCLIC-NUCLEOTIDE PHOSPHODIESTERASE : An Early Defect in Inherited Retinal Degeneration of C3H Mice

Cyclic nucleotides have been implicated in the differentiation and function of the vertebrate retina. In the normal retina of DBA mice, the specific activity of cyclic-nucleotide phosphodiesterase (PDE), with cyclic-AMP as the substrate (cAMP-PDE), increases eightfold between the 6th and 20th postnatal day. Kinetic analysis of retinae from newborn mice reveals a PDE with a single Michaelis constant (K_m) value for cyclic-AMP (low K_m-PDE). After the 6th postnatal day, a second PDE with a high K_m for cyclic-AMP (high K_m-PDE) can be demonstrated. The appearance and increasing activity of the high K_m-PDE coincides with the differentiation and growth of photoreceptor outer segments. Additionally, the high K_m-PDE is shown by microchemical techniques to be concentrated in the photoreceptor cell layer and the low K_m-PDE within the inner layers of the normal retina. In C3H mice afflicted with an inherited degeneration of the photoreceptor layer, the postnatal increase in the specific activity of cAMP-PDE is substantially lower than in the normal retina. The postnatal increase in the specific activity of cAMP-PDE in two regions of the brain of C3H mice is the same as in the normal strain. A deficiency in high K_m-PDE activity in the C3H retina is evident on the 7th postnatal day, when the activity of low K_m-PDE, photoreceptor morphology, and rhodopsin content of these retina are essentially normal. In the adult C3H retina, the PDE activity with cyclic-GMP and cyclic-UMP as substrates is significantly below that of the normal retina. These data indicate that an alteration in cyclic-AMP metabolism occurs before photoreceptor cell degeneration in the retinae of C3H mice.     

Rod photoreceptor ribbon synapses in DBA/2J mice show progressive age-related structural changes

The DBA/2J mouse is a commonly used animal model in glaucoma research. The eyes of DBA/2J mice show severe age-related changes that finally lead to the degeneration of retinal ganglion cells and the optic nerve. Recent electroretinogram studies identified functional deficits, which suggest that also photoreceptor cells are involved in the pathological processes occurring in the DBA/2J mouse retina. In a comparative study, we examined anatomical and molecular changes in the retinae of DBA/2J and C57BL/6 control mice with light and electron microscopy and with PCR analyses. In the retina of the DBA/2J mouse, we found a thinning of the outer plexiform layer, the first synaptic layer in the transfer of visual signals, and age-dependent and progressive degenerative structural changes at rod photoreceptor ribbon synapses. The structural ribbon changes represent a photoreceptor synaptic phenotype that has not yet been described in this animal model of secondary angle-closure glaucoma. Furthermore, genes of the classical complement cascade were upregulated in the photoreceptor cells of aging DBA/2J mice, suggesting a putative link between ribbon synapse degradation and the innate immune system.     

Proteome map of normal rat retina and comparison with the proteome of diabetic rat retina: new insight in the pathogenesis of diabetic retinopathy

We have employed proteomics to establish a proteome map of the normal rat retina. This baseline map was then used for comparison with the early diabetic rat retinal proteome. Diabetic rat retinae were obtained from Dark Agouti rats after 10 wk of streptozotocin-induced hyperglycaemia. Extracted proteins from normal and diabetic rat retinae were separated and compared using 2-DE. A total of 145 protein spots were identified in the normal rat retina using MALDI-MS and database matching. LC-coupled ESI-MS increased the repertoire of identified proteins by 23 from 145 to 168. Comparison with early diabetic rat retinae revealed 24 proteins unique to the diabetic gels, and 37 proteins absent from diabetic gels. Uniquely expressed proteins identified included the HSPs 70.1A and 8, and platelet activating factor. There were eight spots with increased expression and 27 with decreased expression on diabetic gels. Beta catenin, phosducin and aldehyde reductase were increased in expression in diabetes whilst succinyl coA ligase and dihydropyrimidase-related protein were decreased. Identification of such changes in protein expression has given new insights and a more comprehensive understanding of the pathogenesis of diabetic retinopathy, widening the scope of potential avenues for new therapies for this common cause of blindness.     

Alterations in cyclic AMP metabolism associated with photoreceptor cell degeneration in the C3H mouse

Previous studies have shown an abnormality in cyclic nucleotide phosphodiesterase activity in the retina of mice (C3H/HeJ) with an inherited degeneration of the photoreceptor layer. Adenyl cyclase activity and cyclic AMP content have been measured in C3H retina and compared with that in normal retina (DBA/1J) during postnatal maturation, to assess the influence of the mutation upon cyclic AMP metabolism. Adenyl cyclase activity increases normally for the first 7 days of age; thereafter, it becomes greater than normal. Cyclic AMP becomes obviously abnormal after 10 days of age. The elevated levels of cyclic AMP persist in the surviving cells of the inner layers of the adult C3H retina. Adenyl cyclase activity and cyclic AMP content are concentrated in the inner layers of the normal retina, while the photoreceptor layer has only a very low level of enzyme activity and cyclic AMP. The data suggest that the synthesis of cyclic AMP in the inner layers of C3H retina is significantly enhanced, during the period of postnatal development in which the photoreceptor cells have begun to degenerate.     

Generation of cytotoxic T lymphocytes in vitro. VII. Suppressive effect of irradiated MLC cells on CTL response.

Irradiated cells obtained from MLC at the peak of the CTL response caused profound suppression of generation of CTL when added in small numbers at the initiation of primary MLC prepared with normal spleen cells. The inhibitory activity of the MLC cells was not affected by irradiation (1000 rads) but was abolished by treatment with anti-theta serum and complement. The suppression was immunologically specific. The response of A (H-2a) spleen cells toward C3H (H-2k) alloantigens was suppressed by irradiated MLC cells obtained from MLC prepared with A spleen cells and irradiated C3H-stimulating cells, whereas the response of A spleen cells toward DBA/2 (H-2d) alloantigens was affected relatively little. However, if irradiated C3H X DBA/2 F1 hybrid spleen cells were used to stimulate A spleen cells in MLC, addition of irradiated MLC cells having cytotoxic activity toward C3H antigens abolished the response to both C3H and DBA/2 antigens. The response to DBA/2 antigens was much less affected when a mixture of irradiated C3H and DBA/2 spleen cells was used as stimulating cells. Thus, the presence of MLC cells having cytotoxic activity toward one alloantigen abolished the response to another non-cross reacting antigen only when both antigens were present on the same F1 hybrid-stimulating cells. This suppression of generation of CTL by irradiated MLC cells apparently involves inactivation of alloantigen-bearing stimulating cells as a result of residual cytotoxic activity of the irradiated MLC cells. This mechanism may be active during the decline in CTL activity noted in the normal immune response in vivo and in vitro.     

Calcium-binding proteins in the retina of a calbindin-null mutant mouse

Calcium-binding proteins are abundantly expressed in many neurons of mammalian retinae. Their physiological roles are, however, largely unknown. This is particularly true for calcium-modulating proteins (“calcium buffers”) such as calbindin D28k. Here, we have studied retinae of wildtype (+/+) and calbindin-null mutant (–/–) mice by using immunocytochemical methods. Although calbindin immunoreactivity was completely absent in the calbindin (–/–) retinae, those cells that express the protein in wildtype retinae, such as horizontal cells, were still present and appeared normal. This was verified by immunostaining horizontal cells for various neurofilament proteins. In order to assess whether other calcium-binding proteins are upregulated in the mutant mouse and may thus compensate for the loss of calbindin, mouse retinae were also immunolabeled for parvalbumin, calretinin, and a calmodulin-like protein (CALP). In no instance could a change in the expression pattern of these proteins be detected by immunocytochemical methods. Thus, our results show that calbindin is not required for the maintenance of the light-microscopic structure of the differentiated retina and suggest roles for this protein in retinal function.     

The role of TGFβ1 stimulating ROCK I signal pathway to reorganize actin in a rat experimental model of developmental dysplasia of the hip

The aim of the present study was to investigate the protective effects of Trigonella foenum-graecum Linn. (fenugreek) in Streptozotocin-induced diabetic rat retina. Fenugreek (100 and 200 mg/kg body weights) treatment was carried out for 24 weeks and evaluated for inflammatory [tumor necrosis factor (TNF)-α and interleukin (IL)-1β] and angiogenic [vascular endothelial growth factor (VEGF) and protein kinase C (PKC)-β] molecular biomarkers. Retinal oxidative stress was evaluated by estimating antioxidant (Glutathione, Superoxide dismutase, and Catalase) parameters. Fluorescein angiography was performed to detect retinal vascular leakage. Electron microscopy was performed to determine basement membrane thickness. In the present study, significant rises in the expressions of retinal inflammatory (TNF-α and IL-1β) and angiogenic (VEGF and PKC-β) molecular biomarkers were observed in diabetic retinae compared with normal retinae. However, fenugreek-treated retinae showed marked inhibition in the expression of inflammatory and angiogenic molecular biomarkers. Moreover, results from the present study showed positive modulatory effects of fenugreek on retinal oxidative stress. Fluorescein angiograms and fundus photographs obtained from diabetic retinae showed retinal vascular leakage. On the other hand, fenugreek-treated retinae did not show vascular leakage. Further, thickened BM was recorded in diabetic retina compared with normal retinae. However, fenugreek-treated retinae showed relatively lesser thickening of capillary BM. In conclusion, it may be postulated that fenugreek has great potential in preventing diabetes-induced retinal degeneration in humans after regular consumption in the specified dosage.     

Pregnancy rates, prenatal and postnatal survival of offspring, and litter sizes after reciprocal embryo transfer in DBA/2JHd, C3H/HeNCrl and NMRI mice

Success of embryo transfer is often a limiting factor in transgenic procedures and rederivation efforts, and depends on the genetic background of the donor and recipient strains used. Here we show that embryo transfer to DBA/2J females is possible, and present data on pre- and postnatal success rates after reciprocal embryo transfer using the inbred DBA/2J and C3H/HeN, and outbred NMRI strains. The highest embryo yield was achieved in outbred NMRI females, but embryo yields were similar in DBA/2J and C3H/HeN mice following superovulation despite poor estrus cycle synchronization in DBA/2J females. In-strain transfer of DBA/2J blastocysts (transfer of embryos to recipients from the same strain) resulted in pregnancy rates (57.1%) similar to those obtained following in-strain transfer of C3H/HeN (60.0%) and NMRI mice (83.3%), although the prenatal survival rate of blastocysts was low. Moreover, from the pups born only half survived the postnatal period after transfer of DBA/2J and C3H/HeN blastocysts to DBA/2J recipients. These problems were not observed when transferring NMRI-blastocysts to C3H/HeN and DBA/2J mothers. The number of blastocysts transferred also had a positive effect on the success of embryo transfer. In conclusion, C3H/HeN and DBA/2J females can be used as recipients for embryo transfer procedures for certain donor strains like NMRI, as one major determinant seems to be the genetic background of the embryos transferred. We also recommend to increase the number of DBA/2J blastocysts transferred, and to foster the DBA/2J pups to other DBA/2J mothers postnatally for in-strain transfer of DBA/2J mice.     

Dystrophic cardiac calcinosis in mice: abnormal myocardial response to freeze-thaw injury

Dystrophic cardiac calcinosis (DCC) is a frequent finding in DBA/2, C3H and BALB/c mice and its etiology is not known. Previous studies have speculated that myocardial necrosis is involved in the pathogenesis of DCC. In this study, cardiac necrosis was induced in DBA/2, C3H and C57BL/6 mice by freeze-thaw injury through the abdominal diaphragm. Four weeks after freeze-thawing, the mice were sacrificed and the hearts and diaphragms were examined. In response to injury, cardiac mineralization was present only in DBA/2 and C3H mice. The myocardium of C57BL/6 mice (control strain) healed by fibrosis without mineralization, the normal response of the myocardium to injury. Calcified diaphragms also were present at the site of freeze-thaw injury in DBA/2 and C3H mice, which is supportive evidence that a systemic abnormality is involved in the pathogenesis of DCC. The conclusion from this study is that the pathogenesis of DCC in DBA/2 and C3H mice is multifactorial and involves both myocardial necrosis and an abnormal response to injury.     

Comparison of immune responsiveness in mice after single or multiple donor-specific transfusions

Immune responsiveness was compared in B6AF1 mice after one, two, three, or four donor-specific DBA/2 blood transfusions (DST). Ten days after the last transfusion, the spleen cells of transfused mice were assayed for direct lymphocyte-mediated cytotoxicity, for the ability to respond in mixed lymphocyte culture (MLC) and cell-mediated lymphocytotoxic (CML) assays to DBA/2 and C3H/He antigens, and for the ability to inhibit the MLC and CML response of normal B6AF1 to DBA/2 and C3H/He antigens. Immune responsiveness was also tested in B6AF1 2 to 80 days after a single DBA/2 DST. The MLC response of transfused mice was specifically suppressed to the blood donor after both single and multiple transfusions. The CML response to DBA/2 was suppressed after a single DST, but returned to normal after multiple transfusions. Spleen cells from transfused mice did not inhibit the MLC response of normal B6AF1 mice to DBA/2 or C3H/He antigens after one or two transfusions regardless of time tested, but were able to inhibit the response to both stimulators after three or more transfusions. The MLC response remained specifically suppressed to the blood donor for as long as 80 days after a single DST, while the CML response was suppressed up to 50 days after transfusion, but had returned to normal by 80 days.     

A decrease in guanylate cyclase activity in some tissues of C3H/HeJ mice with retinal dystrophy

Guanylate cyclase activity was assayed in homogenates, in particulate and soluble fractions from retina, cerebellum, cerebral cortex and adrenal gland of adult C3H/HeJ mice with a dystrophic retinopathy. In comparison to control mice (DBA/1J), in C3H/HeJ strain a significant decrease in guanylate cyclase activity occurred in homogenates from retina, cerebellum and adrenal gland. In particular a significant decrease was found in particulate fraction of retina, in the soluble fraction of cerebral cortex and cerebellum and in both fractions of the adrenal gland. In contrast to the retina and cerebellum where guanylate cyclase activity in homogenates was found significantly decreased both in the male and female, in the cerebral cortex guanylate cyclase decreased in both sexes although in female this was more marked.     

Synaptic organization of the indoleamine-accumulating neurons in the cyprinid retina

The distribution and synaptic connections of the indoleamine-accumulating neurons in the retinae of the goldfish and carp were studied by means of fluorescence and electron microscopy. The indoleamine-accumulating neurons were visualized after intravitreal injection and uptake of the indoleamine 5,6-dihydroxytryptamine. This labeling procedure produced a characteristic yellow fluorescence of the indoleamine-accumulating neurons and also characteristic ultrastructural changes in these cells. To avoid interference from the dopaminergic neurons of the retina, their processes were either removed by prior treatment with 5-hydroxydopamine or prevented from taking up 5,6-dihydroxytryptamine by the simultaneous injection of the catecholamine alpha-methyl-noradrenaline. Fluorescence-microscopic studies confirmed earlier reports that the indoleamine-accumulating perikarya and processes are distributed similar to those of amacrine cells. The indoleamine-accumulating processes ramify in three bands in the inner plexiform layer, the outermost one being the densest. Electron-microscopic investigations showed the indoleamine-accumulating neurons to have synapses of the conventional type, similar to amacrine cells. Their main synaptic contacts are with other amacrine cells, but synapses with bipolar cell terminals are also present. Both the distribution of the indoleamine-accumulating processes and their synaptic arrangement in the cyprinid retina differ from those found in mammalian retinae investigated previously.     

Genetic influences on oestrous cyclicity in mice: evidence that cycle length and frequency are differentially regulated.

Studies in C57BL/6J, DBA/2J and C3H/HeJ mice and in two F1 hybrid strains (B6D2F1 and B6C3HF1) 2-5 months old revealed marked genotypic differences among inbred strains. C57 mice had three times as many regular (3-6 days) cycles as DBA and C3H mice, due largely to fewer pseudopregnant-like (7-14 day) cycles. C57 had longer regular cycles than DBA and C3H mice. Although the frequencies of regular cycles of DBA and C3H mice were similar, the cycles of C3H mice were shorter than those of DBA mice. The results indicated that the genetic determinants of the frequency of regular cycles differ from those specifying cycle length. Frequency of regular cycles of F1 hybrids was either intermediate between the parent strains (B6D2F1) or similar to the C57 strain (B6C3HF1), suggesting that regular cycle frequency shows additive genetic variation in the former crosses, but mostly dominant variance in the latter background. Regular cycles were either shorter than in both parent strains (B6D2F1) or similar to one of them (B6C3HF1), indicating heterosis and dominance for genes specifying short cycles. Although the lack of reciprocal crosses meant that maternal effects and possible genomic imprinting effects could not be assessed, these results reveal marked genetic influences on cycle length and frequency and suggest that some of the genes specifying these two traits differ.     

Regional variations in the outer retina of atherinomorpha (Beloniformes,Atheriniformes, Cyprinodontiformes: Teleostei): photoreceptors,cone patterns,and cone densities

The outer retinae of adults of 13 atherinomorph species, representing nine different families, were examined by both light and electron microscopy. The retinae were investigated with respect to photoreceptor types, cone densities, and cone patterns. All data were composed to eye maps. This procedure allows an interspecific comparison of the regional differences within the outer retina among these shallow-water fish. Furthermore, for a more detailed pattern analysis nitro-blue tetrazolium chloride- (NBT)-stainings in the retina of Melanotaenia maccullochi are presented. Apart from rods, eight morphologically different cone types could be identified: short, intermediate, and long single cones, double cones (equal and unequal), triple cones (triangular and linear), and in Ameca splendens one quadruple cone. Dimensions and occurrence of photoreceptors vary among the respective species and within the retinal regions. In the light-adapted state, the cones are arranged in highly ordered mosaics. Five different cone tessellation types were found: row patterns, twisted row patterns, square patterns, pentagonal patterns, and, exclusively in Belone belone, a hexagonal pattern. In Melanotaenia maccullochi the different spectral photoreceptor classes correspond well with the distribution of morphological photoreceptor classes within the mosaic. Double cone density maxima together with a highly ordered cone arrangement usually occur in the nasal and/or ventral to ventrotemporal retina. In most of the species that were examined these high-density regions are presumed to process visual stimuli from the assumed main directions of vision, which mainly depend on feeding behavior and predator pressure. Our findings are discussed with respect to the variable behavioral and visual ecology and phylogeny of the respective species.     

Immunoglobulin diversity: analysis of the germ-line VH gene repertoire of the murine anti-GAT response.

A cDNA clone was constructed from a mRNA encoding an anti-GAT (Glu60 Ala30 Tyr10) BALB/c monoclonal antibody heavy chain. Its sequence, covering codons -5 to 162 and therefore encompassing the complete V-D-J region, was determined. Surprisingly, the sequence of the VH gene-encoded region was almost identical with that of the BALB/c VH anti-HNP (4-hydroxy-3-nitrophenyl) acetyl VH region, suggesting that the same VH germ-line might be used to encode two heavy chains contributing to antibodies of discrete specificities. A specific VH probe was derived and annealed to Eco RI and Bg1 II restriction fragments of liver (unrearranged) DNA extracted from the BALB/c, DBA/2 and C57BL/6 mouse strains that differ in their H chain allotypes. Under stringent conditions, only a few bands were identified by Southern blotting. The different patterns observed suggest that the VH anti-GAT repertoire differs between these strains even though their various anti-GAT antibodies express the same public idiotypic specificities.     

The heat-shock response co-inducer arimoclomol protects against retinal degeneration in rhodopsin retinitis pigmentosa

Retinitis pigmentosa (RP) is a group of inherited diseases that cause blindness due to the progressive death of rod and cone photoreceptors in the retina. There are currently no effective treatments for RP. Inherited mutations in rhodopsin, the light-sensing protein of rod photoreceptor cells, are the most common cause of autosomal-dominant RP. The majority of mutations in rhodopsin, including the common P23H substitution, lead to protein misfolding, which is a feature in many neurodegenerative disorders. Previous studies have shown that upregulating molecular chaperone expression can delay disease progression in models of neurodegeneration. Here, we have explored the potential of the heat-shock protein co-inducer arimoclomol to ameliorate rhodopsin RP. In a cell model of P23H rod opsin RP, arimoclomol reduced P23H rod opsin aggregation and improved viability of mutant rhodopsin-expressing cells. In P23H rhodopsin transgenic rat models, pharmacological potentiation of the stress response with arimoclomol improved electroretinogram responses and prolonged photoreceptor survival, as assessed by measuring outer nuclear layer thickness in the retina. Furthermore, treated animal retinae showed improved photoreceptor outer segment structure and reduced rhodopsin aggregation compared with vehicle-treated controls. The heat-shock response (HSR) was activated in P23H retinae, and this was enhanced with arimoclomol treatment. Furthermore, the unfolded protein response (UPR), which is induced in P23H transgenic rats, was also enhanced in the retinae of arimoclomol-treated animals, suggesting that arimoclomol can potentiate the UPR as well as the HSR. These data suggest that pharmacological enhancement of cellular stress responses may be a potential treatment for rhodopsin RP and that arimoclomol could benefit diseases where ER stress is a factor.     

Developmental pattern of cysteine sulphinic acid transaminase activity in some areas of mice nervous system

Cysteine sulphinic acid (CSA), a key intermediate in the major pathway of taurine metabolism, is catabolised by cysteine sulphinic acid decarboxylase (CSA-D) to hypotaurine and by the cysteine sulphinic acid transaminase (CSA-T) to glutamate. To continue earlier studies about the latter enzyme, the CSA-T activity was measured in the developing olfactory bulb, cerebellum, whole brain and retina of C57/B16 and C3H/He inbred mice. Differences in the developmental pattern between these two strains of mice in the retinae and in the olfactory bulb were observed. The significance of the differences is discussed.