Biomedical subjects

Cassandra L Schlamp

Publications and source records attributed to Cassandra L Schlamp.

6 recordsLinked to original sources

PubMed2006

Decrease of cone opsin mRNA in experimental ocular hypertension.

PURPOSE: This study was designed to test the hypothesis that photoreceptors are adversely affected in glaucoma. As a measure of this effect, we examined the levels of rod opsin, and red/green and blue cone opsin mRNAs in monkeys with experimental ocular hypertension and glaucoma and in human eyes from donors with diagnosed glaucoma. METHODS: Experimental ocular hypertension was induced in one eye of 19 cynomolgous and 2 rhesus monkeys by laser ablation of the trabecular meshwork. In 15 monkeys, the elevated IOP was reduced by trabeculectomy. When the animals had experienced prolonged elevations of IOP (128 to 260 days), they were killed and the eyes enucleated. Fresh retinal tissue from the macula, inferotemporal retina (mid-peripheral), and far peripheral regions were harvested from some animals using a 3 mm trephine. The remaining retinas from these monkeys, and whole retinas from other animals were fixed. RNA isolated from each trephined sample was used for RNase Protection Analysis or real time PCR analysis to quantify opsin mRNA levels from different photoreceptor cell types. Fixed tissue was used for in situ hybridization studies. Human donor eyes (7 glaucoma and 4 control) were obtained from eye banks. All human specimens were used for in situ hybridization studies. RESULTS: Quantitative mRNA analysis and in situ hybridization studies both showed a reduction in the expression of red/green and blue cone opsin mRNAs in 6 monkey eyes with chronic ocular hypertension, relative to the contralateral eye. No loss of rod opsin mRNA was observed. The principal reduction occurred in cells of the mid-peripheral retina, a region of retina that often shows early and progressive damage in humans with glaucoma. In monkeys with ocular hypertension followed by trabeculectomy, there was a similar decrease in cone opsin mRNAs, but only in six out of fifteen (40%) of the monkeys. The decrease in these animals was correlated with a significantly elevated IOP at some time during the 2 weeks prior to euthanization and not with the extent of glaucomatous damage. Of the 7 human eyes with diagnosed glaucoma that were examined, 5 showed a decrease of cone opsin mRNA in the mid-peripheral retina, whereas none of the 4 normal eyes examined showed a decrease. CONCLUSIONS: Ocular hypertension leading to glaucoma also affects the outer retina, particularly the cone photoreceptors. We speculate that these cells become stressed leading to a disruption in the expression of normal genes, such as that encoding opsin. There is some evidence that this effect is reversible, when IOP levels are reduced.

Aged↗

PubMed2006

Progressive ganglion cell loss and optic nerve degeneration in DBA/2J mice is variable and asymmetric.

BACKGROUND: Glaucoma is a chronic neurodegenerative disease of the retina, characterized by the degeneration of axons in the optic nerve and retinal ganglion cell apoptosis. DBA/2J inbred mice develop chronic hereditary glaucoma and are an important model system to study the molecular mechanisms underlying this disease and novel therapeutic interventions designed to attenuate the loss of retinal ganglion cells. Although the genetics of this disease in these mice are well characterized, the etiology of its progression, particularly with respect to retinal degeneration, is not. We have used two separate labeling techniques, post-mortem DiI labeling of axons and ganglion cell-specific expression of the betaGeo reporter gene, to evaluate the time course of optic nerve degeneration and ganglion cell loss, respectively, in aging mice. RESULTS: Optic nerve degeneration, characterized by axon loss and gliosis is first apparent in mice between 8 and 9 months of age. Degeneration appears to follow a retrograde course with axons dying from their proximal ends toward the globe. Although nerve damage is typically bilateral, the progression of disease is asymmetric between the eyes of individual mice. Some nerves also exhibit focal preservation of tracts of axons generally in the nasal peripheral region. Ganglion cell loss, as a function of the loss of betaGeo expression, is evident in some mice between 8 and 10 months of age and is prevalent in the majority of mice older than 10.5 months. Most eyes display a uniform loss of ganglion cells throughout the retina, but many younger mice exhibit focal loss of cells in sectors extending from the optic nerve head to the retinal periphery. Similar to what we observe in the optic nerves, ganglion cell loss is often asymmetric between the eyes of the same animal. CONCLUSION: A comparison of the data collected from the two cohorts of mice used for this study suggests that the initial site of damage in this disease is to the axons in the optic nerve, followed by the subsequent death of the ganglion cell soma.

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PubMed2004

Insertion of the beta Geo promoter trap into the Fem1c gene of ROSA3 mice.

ROSA3 mice were developed by retroviral insertion of the beta Geo gene trap vector. Adult ROSA3 mice exhibit widespread expression of the trap gene in epithelial cells found in most organs. In the central nervous system the highest expression of beta Geo is found in CA1 pyramidal cells of the hippocampus, Purkinje cells of the cerebellum, and ganglion cells of the retina. Characterization of the genomic insertion site for beta Geo in ROSA3 mice shows that the trap vector is located in the first intron of Fem1c, a gene homologous to the sex-determining gene fem-1 of Caenorhabditis elegans. Transcription of the Rosa3 allele (R3) yields a spliced message that includes the first exon of Fem1c and the beta Geo coding region. Although normal processing of the Fem1c transcript is disrupted in homozygous Rosa3 (Fem1c(R3/R3)) mice, some tissues show low levels of a partially processed transcript containing exons 2 and 3. Since the entire coding region of Fem1c is located in these two exons, Fem1c(R3/R3) mice may still be able to express a putative FEM1C protein. To this extent, Fem1c(R3/R3) mice show no adverse effects in their sexual development or fertility or in the attenuation of neuronal cell death, another function that has been attributed to both fem-1 and a second mouse homolog, Fem1b. Examination of beta Geo expression in ganglion cells after exposure to damaging stimuli indicates that protein levels are rapidly depleted prior to cell death, making the beta Geo reporter gene a potentially useful marker to study early molecular events in damaged neurons.

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PubMed2003

Vitamin D analogues increase p53, p21, and apoptosis in a xenograft model of human retinoblastoma.

PURPOSE: To study the antineoplastic effect of vitamin D analogues in a xenograft model of human retinoblastoma. METHODS: Athymic mice were injected subcutaneously with Y79 cells and treated 5 days a week with either mineral oil (control group) or the vitamin D analogues calcitriol or 1,25-dihydroxy-16-ene-23-yne vitamin D(3) (16,23-D(3)). BrdU was injected 1 hour before death. Animals were killed after 1, 2, 3, or 5 weeks. Paraffin-embedded sections of the tumors were studied for cell proliferation by monitoring for BrdU incorporation and cell death by terminal transferase dUTP-nick end labeling (TUNEL), 3'-overhang ligation, and histology. Sections of the tumors were immunostained for p53 and p21. RESULTS: There was no significant difference in incorporation of BrdU among the three groups, suggesting that cell proliferation is unaffected by vitamin D analogues. TUNEL was increased in tumors treated with vitamin D analogues compared with the control group. This increase was statistically significant for calcitriol in the time frame examined, but not statistically significant for 16,23-D(3). Alternatively, the ratio of proliferation to cell death was significantly different for both calcitriol and 16,23-D(3) compared with control tumors after 3 weeks of treatment. Dying cells contained DNA strand breaks with overhanging nucleotides and nuclear changes characteristic of apoptosis. There was an increase in staining for p53 and p21 in areas associated with cell death in specimens treated with vitamin D analogues. CONCLUSIONS: Vitamin D analogues appear to attenuate retinoblastoma tumor growth in athymic mice by increasing apoptosis. Cell death is associated with the upregulation of both p53 and p21.

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PubMed2002

p53 regulates apoptotic retinal ganglion cell death induced by N-methyl-D-aspartate.

PURPOSE: The tumor suppressor protein p53 plays a central role in regulating apoptosis in a variety of neuronal cell types. Previous studies have indicated that retinal ganglion cell (RGC) death induced by ischemia follows a p53-dependent pathway. Ischemia causes wide-spread damage to the retina, eliciting multiple different damaging pathways. We conducted experiments to specifically investigate the role of p53 in RGC death activated by overstimulation of the N-methyl-D-aspartate (NMDA) receptor, an ionotropic glutamate dependent calcium channel normally involved in glutamate neurotransduction. METHODS: RGC death was induced in both wild-type (CB6F1 or 129/Sv) and p53-deficient (129/Sv background) mice by a single intravitreal injection of either 40 or 160 nmol of NMDA into one eye leaving the other eye as an untreated control. Cell loss was quantified by comparing the number of surviving cells in the retinas from experimental eyes relative to the control eyes of the same animals. The accumulation of p53 mRNA in retinas was monitored by reverse-transcription PCR (RT-PCR) of retinal total RNA isolated from mice injected with 40 nmol of NMDA. The functional requirement for p53 was monitored in p53-deficient mice after intravitreal injection of 160 nmol of NMDA. Immunohistochemistry for cleaved poly(ADP-ribose) polymerase (PARP) was performed on p53-deficient mice after intravitreal injection of 160 nmol of NMDA. RESULTS: In wild-type CB6F1 mice, p53 mRNA levels are elevated within 3 h after NMDA injection. This accumulation correlates with the onset of changes in RGC nuclear morphology that precedes pyknosis, which occurs by 6 h. Mice (129/Sv) deficient for one or both alleles of p53 show no developmental change in RGC number, compared to wild-type animals (Mann-Whitney test, p=0.824), suggesting that p53 is not required for developmental programmed cell death of RGCs. In adult mice, however, p53-dependent changes in the rate of RGC death after exposure to 160 nmol of NMDA were observed. Four days after injection, p53+/+ and p53-/- mice exhibit statistically equivalent amounts of cell loss (p>0.1), while p53+/- mice have significantly attenuated cell loss (p<0.002), relative to the other groups. RGCs from NMDA-treated p53+/+ and p53-/- mice were analyzed further using immunohistochemistry to identify the cleavage products of poly(ADP-ribose) polymerase (PARP), a known substrate for caspases. Cleaved PARP was found in p53+/+ and p53+/- eyes, but not in p53-/- mice. CONCLUSIONS: Developmental RGC programmed cell death does not require p53. Selective overstimulation of the glutamate-dependent NMDA-receptor in adult mice activates a p53-dependent pathway of death in RGCs. The requirement for p53 is not absolute, however, because mice lacking this gene are able to execute an alternative pathway of cell death. Examination of the cleavage of PARP, which is a substrate for caspases, suggests that the p53-dependent pathway utilizes these proteases, but the p53-independent pathway does not.

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PubMed2002

Vitamin D analogs, a new treatment for retinoblastoma: The first Ellsworth Lecture.

PURPOSE: This lecture honors the memory of Dr. Robert M. Ellsworth, an important figure in the development of current treatments of retinoblastoma (RB), and reviews our studies of vitamin D analogs as treatments for retinoblastoma in two experimental mouse models. We identified vitamin D receptors in retinoblastoma and examined the effectiveness and mechanism of action of these analogs. METHODS: Reverse-transcriptase polymerase chain reaction (RT-PCR) amplification was used to detect vitamin D receptor mRNAs in human and mouse retinoblastomas. The effectiveness and toxicity of vitamin D(2), calcitriol, and synthetic analogs were studied in the athymic/Y-79 xenograft and transgenic mouse models of RB. Dosing was 5X/week for five weeks. Dose-response studies focused on tumor inhibition; toxicity studies investigated survival and serum calcium. The mechanism of action of vitamin D was investigated using terminal transferase dUTP labeling 3'-overhang ligation to measure apoptosis; immunohistochemistry measured p53-dependent gene expression and cell proliferation. RESULT: Vitamin D receptor mRNAs were detectable in Y-79 RB cells, LH beta-Tag tumors, and human RB specimens using RT-PCR. Calcitriol inhibited cell growth in vitro. Calcitriol and vitamin D(2) inhibited in vivo growth in xenograft and transgenic models, but therapeutic levels were toxic due to hypercalcemia. Two analogs, 16,23-D(3) and 1 alpha-OH-D( 2), inhibited tumors in animal models of RB with reduced toxicity. The mechanism of action appears related to increased p53-related gene expression resulting in increased apoptosis. CONCLUSION: 16,23-D(3) and 1 alpha-OH-D(2) are effective in tumor reduction in two mouse models of RB with low toxicity. These results warrant initiating phase 1 and phase 2 clinical studies in children.

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