Biochemical evidence for conversion to milder form of hereditary mouse cataract by different genetic background

Congenic hereditary cataract mice, BALB/c-nct/nct, were established by introducing the nct gene from Nakano into BALB/c mice. These mice developed a milder cortical form of cataract which developed sporadically and later in life than in Nakano mice. Combined use of BALB/c and BALB/c-nct/nct mice enables biochemical comparison of normal clear lenses, congenic clear lenses which are destined to be opacified some time later, and opacified lenses in the same genetic and aging statuses. We compared the age-related changes in water content and water-soluble and -insoluble fractions among these three types of lenses. Congenic clear lenses and opaque lenses were more similar to BALB/c normal clear lenses and Nakano opaque ones, respectively, in these parameters. These results suggest, in addition to formation of aggregated crystallins and their accumulation in water-insoluble fractions, that decreased protein synthesis, increased protein degradation and augmented leakage of crystallin might have a significant role in the nct-induced lens opacification.     

Genetic analysis of Nakano Cataract and its modifier genes in mice

The Nakano Cataract (NCT) is an autosomal, recessive, single gene mutation in mice leading to an osmotic cataract induced by an endogenous inhibitor of Na, K-ATPase. In this report, we further refined the map position of the mutant locus to a <0.7c M segment between D16Mit5 and D16Mit185 in 1,000 BALB/c-nct/nct x(BALB/c- nct/nctxMSM)F1 backcrossed mice with PCR-based microsatellite analysis. The NCT in the original Nakano mice developed at 3 weeks of age, rapidly formed a pin-head type dense opacity, whereas the cataract in the congenic BALB/c- nct/nct mice developed at 5-6 weeks of age or later, slowly formed a diffuse opacity. A major histological difference was the presence or absence of heavy condensation of the lens nucleus. These two types of cataract were segregated in the backcrossed mice. Linkage analysis of the two subtypes among the backcrossed mice revealed two recessive BALB/c-derived modifier genes on chromosome 3 and 10.     

Age-related changes of calpain II and alpha-crystallin in the lens of hereditary cataract (Nakano) mouse

The age-related changes of calpain II (high-Ca2+-requiring form of Ca2+-dependent cysteine proteinase; EC 3.4.22.17) and alpha-crystallin in the lens of hereditary cataract (Nakano; cac/cac) mouse were studied. Before the onset of the cataract formation, i.e., at the end of the 2nd week after birth, the calpain activity in Nakano mice was as high as that in the control ICR mice, but it decreased rapidly as the cataract progressed to completion during the 4th and the 12th week. Marked degradation of lens proteins ensued between the 2nd and the 4th weeks, and one of these proteins was identified, using monospecific antibodies, as B chain of alpha-crystallin. A chain of alpha-crystallin was not degraded in vivo, in contrast to its known susceptibility to calpain in vitro. The present data suggest that in Nakano mice, calpain may be involved in the onset or early stage of the cataract formation.     

Age and cataract-related changes in the heavy molecular weight proteins and gamma crystallin composition of the mouse lens.

Heavy molecular weight (HMW) proteins were detected in normal and cataractous mouse lenses. The HMW aggregates increased with the age of the lens in normal mouse. Alpha and β-crystallins were detected by immunodiffusion in the HMW fractions from normal and Nakano mice. No γ-crystallin could be detected in these aggregates by immunodiffusion; however, a slight amount of this crystallin was detected using the radioimmunoassay. The polypeptide composition of the HMW proteins was different in the Nakano mouse from the normal. By SDS polyacrylamide gel electrophoresis, a polypeptide of 27 000 mol. wt. was evident in the Nakano HMW material that was not present in the normal HMW protein, but a 15 000 mol. wt. band was absent in the Nakano.Two other differences were seen with the Nakano lens. First, the water insoluble lens protein was extremely high. By 90 days, about two thirds of the protein was insoluble in these lenses. Secondly, the sharp drop in γ-crystallin at the time of complete opacification of the lens was in part a result of the leakage of this protein into the anterior chamber of these mice. By radioimmunoassay, the level of γ-crystallin in the Nakano aqueous humor at the time of the cataract was greater than 100 ng per microliter. These data demonstrate that crystallins are converted to the insoluble proteins and some diffuse out of the lens during cataract formation.     

Genetic background determines susceptibility to experimental immune-mediated blepharoconjunctivitis: comparison of Balb/c and C57BL/6 mice

Balb/c and C57BL/6 mice have been reported to be biased towards Th2 and Th1 immune responses, respectively. We investigated which strain is more susceptible to the development of experimental immune-mediated blepharoconjunctivitis (EC), which is predominantly mediated by Th2 immune responses. EC was induced by three different methods in Balb/c and C57BL/6 mice using ragweed (RW) as the antigen. The mice were thus either actively immunized with RW, passively immunized by transfer of RW-primed T cells, or passively immunized by transfer of RW-specific IgE, followed by RW challenge in eye drops. Twenty-four hours after the challenge, conjunctivas, sera and spleens were harvested for histological analysis, measurement of serum IgE and assessment of cellular immune responses, respectively. The responses of the Balb/c and C57BL/6 mice were compared. In addition, to assess the involvement of IFN-gamma in the development of EC in the two strains, IFN-gamma knockout (GKO) mice of the two strains were actively immunized and evaluated as above. Regardless of the method of induction, EC, as determined by the degree of eosinophil infiltration into the conjunctiva, was more severe in Balb/c mice than in C57BL/6 mice. Moreover, more IgE was produced by actively immunized Balb/c mice than C57BL/6 mice and RW-primed splenocytes from Balb/c mice produced more IL-4 but less IFN-gamma than those from C57BL/6 mice. EC could be induced in the GKO mice of both strains. However, when their EC was compared to that in WT mice, significantly less infiltration of eosinophils was noted in the Balb/c GKO mice. Taken together, Balb/c mice are more susceptible to EC than C57BL/6 mice and this higher susceptibility might be related to the Th2 immune response bias of Balb/c mice. Furthermore, the involvement of endogenous IFN-gamma in the development of EC in these two strains differs.     

Stimulation of the hexose monophosphate pathway by pyrroline-5-carboxylate reductase in the lens

Addition of pyrroline-5-carboxylate (P5C) or its precursors to rat lenses cultured for 24 hr in TC-199 medium containing 14C-glucose results in an apparent concentration-dependent increase in hexose monophosphate-pentose (HMP) pathway activity. Addition of proline, the reduction product of P5C, did not result in an increase, suggesting that stimulation of the HMP pathway is related to the reduction of P5C to proline by the enzyme P5C reductase. No apparent feedback inhibition on P5C reductase was observed. Stimulation of HMP pathway activity by P5C was also observed in the lenses of Philly and Nakano mouse, two models of congenital osmotic cataracts. Compared with its genetic control, the Swiss--Webster mouse, generally no difference in the lenticular levels of HMP pathway activity was observed in the Philly mouse--even after the onset of cataract. Stimulation of the HMP pathway in the Philly lens by P5C, however, was consistently lower than its control. In the lenses from the Nakano mouse and its genetic control, the Balb/c mouse, no difference in the percentage stimulation of the HMP pathway resulting from the addition of P5C was observed, but HMP pathway activity in the Nakano lens was consistently lower than that of the control.     

Biosynthesis of proteoglycans by lens epithelial cells of cataractous mouse (Nakano strain)

The capsules (with epithelial cells attached) of lenses from normal and cataractous mice (Nakano strain) were biosynthetically labeled in vitro with radioactive precursors. The labeled macromolecules were chromatographed on a Sepharose CL-4B column and analyzed by specific enzyme digestion. The incorporation of [3H]-proline and [3H]-glucosamine into macromolecules was comparable in the cataract and normal capsules, while that of [35S]-sulfate was reduced by 60% in the cataract capsules, indicating that the proteoglycan synthesis was specifically decreased in the cataract lens. Glycosaminoglycan analyses showed an increased synthesis of hyaluronic acid and decreased synthesis of heparan sulfate in the cataract capsules. It is possible that the alterations in the synthetic level and glycosaminoglycan components of proteoglycan affect the permeabilities of macromolecules to lens capsule and lead to cataract in Nakano mouse lens.     

Studies on primary cultures of adult lens cells from normal and hereditary cataractous mice.

Lens epithelial cells from normal and congenital cataractous mice strains were cultured under similar conditions. Both normal and cataractous cells actively propagated and reached confluency on the eleventh day. These cells, thereafter, underwent morphological changes characterized by cell elongation, aggregation and formation of lentoid bodies at about 15 days.Electron microscopy revealed these lentoid bodies to consist of immature lens cells. These structures derived from cataractous cells had numerous vacuoles in the cytoplasm much more so than in the normal lens cells. In addition, some lentoid bodies closely resembled mature fibers of the intact lens. It was also demonstrated that these lentoid bodies showed positive immunofluorescence when reacted with fluorescent antiserum to γ-crystallin.There were certain differences observed between the cultured cells derived from normal lens and Nakano cataract. The disappearance of organelles and denucleation process were delayed in the lentoid bodies found in cultured Nakano cells when compared to normal cell culture. In addition a second type of lentoid body, although present as a minor population, was observed in the Nakano cell culture. Other subtle differences were observed during the course of culturing normal and cataractous lens cells.     

Comparative structural and functional analysis of photoreceptor neurons of Rho-/- mice reveal increased survival on C57BL/6J in comparison to 129Sv genetic background

To explore the possible influence of defined genetic backgrounds on photoreceptor viability and function in mice carrying a targeted disruption of the rhodopsin gene, the severities of retinopathies in Rho-/- mice on C57BL/6J and 129Sv congenic backgrounds were compared by light microscopy and electroretinography and qualitatively by in situ end labeling of DNA in apoptotic photoreceptor nuclei of retinal sections. Cone photoreceptor viability and function were shown to deteriorate more slowly on the C57BL/6J background in comparison to that of the 129Sv, with significantly greater numbers of outer nuclear layer nuclei in the retinas of C57BL/6J mice at 3 and 4 months of age. Both amplitude and waveform features of the ERG were shown to be remarkably different in the two strains, indicating an approximately 6-fold difference in C57BL/6J Rho-/- mice compared to 129Sv Rho-/- mice at 80 days. Thus, in comparison with the 129Sv strain, genetic modifiers appear to constitute a component of the C57BL/6J background, the expression of which significantly protects cone photoreceptors from apoptotic death in a mutation-induced murine retinopathy. The differences in phenotype revealed in this study are sufficient in principle to provide a basis for comparisons to be made between QTLs in light-induced and mutation-induced systems.     

Hereditary cataract of the Nakano mouse

The Nakano mouse is a hereditary cataract model whose most characteristic change is a deficiency in lens Na+,K(+)-ATPase. Consequently, there is a change in lenticular sodium and potassium ion levels just before cataract formation. The amounts of calcium ion also change suddenly in the lens, with accumulated levels higher than any other type of cataract. Other biochemical changes coincide with the development of lens opacity, including decreases in the levels of reduced glutathione, ATP, biosynthetic activity of proteoglycans in epithelial cells, and the permeability of gap junction channels in fiber cells. The decrease in the activity of Na+,K(+)-ATPase results in changes in a number of key metabolic parameters, resulting in the eventual opacification of the Nakano mouse lens at approximately 30 days of age.     

Immunopathological analysis of T and non-T cell involvement in corneal inflammation caused by HSV type-1 infection

In herpetic keratitis, the role of T cell subsets is not clearly understood although the involvement of cell-mediated immunity has been reported. The purpose of this paper is to analyse T cell-mediated immunopathological conditions and the role of non-T cell lineage in herpetic stromal keratitis employing nude (nu/nu) and Balb/c mice. All mice were divided into three groups. Group 1: Herpes simplex virus type 1 (HSV-1)-inoculated nu/nu mice. Group 2: HSV-1-inoculated nu/nu mice with interleukin-2 (IL-2). Group 3: HSV-1-inoculated Balb/c mice. The conditions of the disease were compared in those three groups clinically and by using histological and immunopathological staining. Following antibodies were employed: anti-Asialo GMI (natural killer). anti-Thy 1 (pan-T). anti-L3T4 (helper/inducer), anti-Lyt 2 (suppressor/killer), anti-Mac 3 (macrophage), anti-Mice Immunoglobulins (monocyte. B.). Corneal lesions showed a reduced inflammatory tendency in nu/nu mice. Topical application of IL-2 in nu/nu mice induced moderate stromal inflammation compared with nu/nu mice. However, the severity of these was less than in Balb/c. Many natural killer cells (NK) were found in nu/nu mice but few in Balb/c mice. These results confirm the involvement of T-cell mediated immunity in stromal inflammation and indicate that NK cell may have an important role in suppressed T-cell immunity in nu/nu mice but are not involved in stromal inflammation.     

Investigation of Nakano lens proteins

Changes in the protein chemistry of the Nakano lens with age and developing cataract and comparison with normal mouse lens protein are reported. It was found that significant differences exist between the protein of the normal and the cataractous lens. In Nakano lenses high molecular weight disulfide-linked aggregates, disulfide-linked cytosol polypeptides to the fiber membrane, an apparent increase in the concentration of degraded polypeptides, disulfide crosslinking of low molecular weight species and marked differences in membrane polypeptide profiles were observed. A striking similarity was found between these observations with the Nakano cataract and previous reports of the changes in protein chemistry in the development of senile human cataract. It can be concluded that although the initiating event for induction of the cataract may differ, the sequence of events following such insult may be similar.     

Ocular lesions in mice following intracerebral injection of herpes simplex virus type I

A clinical isolate of type I Herpes virus was injected intracerebrally in 4-week-old Balb/C mice. Bilateral ocular disease was observed initially clinically as a leukocoria and an anterior uveitis on the 7th to 11th postinjection days. By day 21 an organized vascularized retrolental membrane had formed with resolution of active inflammation and secondary cataract formation. Light microscopy revealed the process to involve a necrotizing retinitis with associated optic nerve demyelination. Electron microscopy and tissue culture demonstrated the virus in involved tissues.     

Biochemical alterations of the lens capsule in mice with hereditary cataract

Lens capsules from normal and cataractous mice (Nakano strain) were compared histologically and chemically. Histologically, the anterior capsule of the cataract lens was thicker than the normal, while the posterior capsule appeared almost the same. Amino acid analysis revealed some differences between normal and cataractous lens capsules: more glycine, hydroxylysine and arginine and less hydroxyproline and tyrosine were found in cataractous capsules. Hydroxyproline showed the largest difference, cataract lens being about 11% lower than normal.To see if the content of hydroxylysine-linked carbohydrates is different in normal and cataractous lenses, as has been shown for renal basement membranes from normal and diabetic animals, a procedure was developed to separate basic amino acids, including the hydroxylysine-linked carbohydrates, from other amino acids. It was found that the content of hydroxylysine-linked disaccharide (Glc-Gal-Hyl) was slightly but significantly higher in cataractous lenses.The possibility was suggested that the compositional alterations observed for the cataract lens might be correlated with functional changes in the lens capsule of the cataract, e.g. lowered elasticity, decreased permeability etc.     

Morphological study on cataractogenesis of the Nakano mouse lens

· Background: Although some histopathological features on the Nakano mouse lens have been pointed out by a few investigators, there seem to have been no detailed studies on the sequential changes that occur. · Methods: We used the following two approaches: (1) Observation of the whole lens by dissection microscopy and (2) light and electron microscopic examination of the sectioned lens specimen. · Results: (1) The Nakano mouse lens showed sustained transparency up to 19 days after birth, fine opacity at the 20th day, and development of a mature cataract around the 30th day. In addition, although the Y-shaped posterior suture was normal at the 15th day, bending of the suture line appeared around the 19th day. (2) The cataractous lens revealed degeneration of the epithelial cells and adjacent anterior cortical fibers at the 10th day. Swelling of the anterior cortical fibers became prominent, and swelling of the posterior cortical fibers occurred by the 15th day. Upon separation of the suture around the 20th day, fine opacity occurred in the perinuclear zone, which extended to the anterior cortex and finally led to the formation of a mature cataract. · Conclusions: These results indicate that epithelial degeneration is a major feature of cataract in the Nakano mouse, and the subsequent lens fiber swelling and posterior sutural separation are the underlying causes of the development of opacity. Received: 2 April 1998 Revised version received: 15 June 1998 Accepted: 23 July 1998     

Lens growth in the Nakano mouse.

The growth curve of the lens of the Nakano mouse was compared to that of the normal mouse. There was no obvious difference for the first two weeks of age. After this period the growth of the normal lens continued while that of Nakano mouse lens slowed abruptly and eventually ceased. Studies with labeled leucine, however, showed that even after the appearance of the "pin-head" opacity the protein synthesis, although depressed, continued. These findings combined with previous histological observations suggest that new fiber formation is probably unaffected in the early stages of the Nakano cataract. The apparent cessation of lens growth is probably associated with the extensive liquefaction that is observed to occur at the posterior nuclear region.     

Changes in lens proteins induced at the early stage of cataractogenesis in cac (Nakano) mice

Changes in lens proteins induced at the early stages of cataractogenesis in cac (Nakano) mice were investigated by two-dimensional gel electrophoresis (2D-PAGE). The 2D-PAGE profile of lens proteins in 21-day-old cac mice differed from that in 27-day-old normal mice, even though the appearance of 'pin-head' nuclear opacity (26-day-old) had not yet been observed in the lenses. Especially noticeable were great differences in the polypeptides associated with the alpha- and beta- crystallin subfractions, the appearances of which corresponded to an increase in a ratio of the amounts of Na+ to that of K+ in the lenses of defective mice. No dramatic decrease in the gamma-crystallin fraction was observed until the mature cataract stage.     

Swelling of the lens fibers.

Lens cells of congenital mouse cataract (Nakano and Fraser strains) and galactose-fed rats were studied by scanning electron microscopy. Similarly lens cells of normal young mice and rats were examined as controls. Normal lenses of young rodents consist of lens fibers in all maturation stages which have been demonstrated in humans and in monkeys. Lens cells in the congenitally cataractous lenses are irregular in size and shape in the earlier stage of the cataract formation. Swelling of the lens cell occurs corresponding to the occurrence of early optical opacity. Swelling of the cell occurs segmentally in congential cataractous lenses; in the apical ends (Fraser) and in the posterior ends (Nakano). Similar swelling of the lens cell is observed in the main cell body in the superficial lens cortex of galactose-fed rats. However, numerous intercellular cysts are formed by the accumulation of fluid which may have been pumped out of the cells before the cells became degenerative. These numerous shrunken fibers are present among swollen cells in both congenital and galactose-induced cataractous lenses.     

Structure-function analysis of rods and cones in juvenile,adult, and aged C57bl/6 and Balb/c mice

To determine whether the photoreceptors change structurally and functionally during aging, and to analyze whether pigmentation in the retinal pigment epithelium might be a contributing factor. Young, adult, and aged C57BL/6 and Balb/c mice (1, 4, and 17 months of age) were housed under a 12-h light/12-h dark cycle, with an ambient light intensity at the eye level of the mice of 85 +/- 18 lux. Scotopic single-flash and photopic-flicker electroretinograms (ERGs) after complete dark adaptation were used to assess rod and cone function, respectively. Numbers of rod photoreceptors were counted in plastic sections, and rhodopsin levels were measured using absorption difference spectrophotometry. Numbers and types of cones were determined using lectin staining in retinal flatmounts and cone-specific antibodies in radial frozen sections. Young pigmented C57BL/6 and nonpigmented Balb/c mice had similar numbers of rods. In both mouse strains, there was an overall decline in rod photoreceptor number during aging, which was more pronounced in albino mice. Rod cell numbers correlated with a drop in the overall amount of rhodopsin and a reduction in the maximum a-wave of the rod ERG. The number of short-wavelength cones was unaffected by age and pigmentation, whereas an age-related decline was observed in mid-wavelength (MWL) cones in albino, but not in pigmented mice. In contrast, MWL cone function was reduced during aging in both strains. Flicker-fusion frequency was determined to be approximately 10 Hz lower in albino animals, which is due to prolonged b-waves in these ERGs. Age-related changes were found in both photoreceptor systems, rods and cones, and in both pigmented and nonpigmented mice. However, rod photoreceptors appear to be more susceptible to both aging and the lack of pigmentation, when compared to cones. These results may help as we begin to understand certain age-related retinal diseases.     

Quantitative trait locus mapping for age-related cataract severity and synechia prevalence using four-way cross mice

PURPOSE: The goal of this study was to map mouse quantitative trait loci (QTL) that influence the development of murine age-related cataract and synechia, by using a genetically heterogeneous mouse population bred by a four-way cross. METHODS: The test population consisted initially of 510 mice bred as the progeny of (BALB/cJ x C57BL/6J)F1 females and (C3H/HeJ x DBA/2J)F1 males. Each mouse was examined by slit lamp at 18 and 24 months of age and scored for degree of lens opacity on a 0 to 4+ scale, and the presence or absence of additional anterior chamber disease was noted. The presence of synechia was confirmed by histology. Each mouse was genotyped at 96 maternal and 92 paternal loci, and the significance of association between genotype and eye lesions was tested by permutation analysis. RESULTS: Significant QTL with effects on lens opacity at 24 months were detected on mouse chromosomes 4, 11, and 12. The effects were additive, and severe cataracts were seen in 80% of the mice with all three high-risk alleles, but in only 28% of the mice with all three low-risk alleles. The risk of synechia was associated with paternal chromosome 1 and on both the maternal and paternally inherited chromosome 4. Mice with all three high-risk alleles had a 68% risk of synechia, compared with a 0% incidence in mice with all three counteralleles. CONCLUSIONS: A four-way cross population of mice can be used to map polymorphic loci that influence cataract severity and synechia prevalence in late life. The results provide a first step toward identification of the individual genes involved and may help to guide the search for homologous human genes.