Effects of brain-derived neurotrophic factor on dopaminergic function and motor behavior during aging.

Brain-derived neurotrophic factor (BDNF) is critical in synaptic plasticity and in the survival and function of midbrain dopamine neurons. In this study, we assessed the effects of a partial genetic deletion of BDNF on motor function and dopamine (DA) neurotransmitter measures by comparing Bdnf(+/-) with wildtype mice (WT) at different ages. Bdnf(+/-) and WT mice had similar body weights until 12 months of age; however, at 21 months, Bdnf(+/-) mice were significantly heavier than WT mice. Horizontal and vertical motor activity was reduced for Bdnf(+/-) compared to WT mice, but was not influenced by age. Performance on an accelerating rotarod declined with age for both genotypes and was exacerbated for Bdnf(+/-) mice. Body weight did not correlate with any of the three behavioral measures studied. Dopamine neurotransmitter markers indicated no genotypic difference in striatal tyrosine hydroxylase, DA transporter (DAT) or vesicular monoamine transporter 2 (VMAT2) immunoreactivity at any age. However, DA transport via DAT (starting at 12 months) and VMAT2 (starting at 3 months) as well as KCl-stimulated DA release were reduced in Bdnf(+/-) mice and declined with age suggesting an increasingly important role for BDNF in the release and uptake of DA with the aging process. These findings suggest that a BDNF expression deficit becomes more critical to dopaminergic dynamics and related behavioral activities with increasing age.

Self-complementary AAV-mediated gene therapy restores cone function and prevents cone degeneration in two models of Rpe65 deficiency.

To test whether fast-acting, self-complimentary (sc), adeno-associated virus-mediated RPE65 expression prevents cone degeneration and/or restores cone function, we studied two mouse lines: the Rpe65-deficient rd12 mouse and the Rpe65-deficient, rhodopsin null ('that is, cone function-only') Rpe65(-/-)::Rho(-/-) mouse. scAAV5 expressing RPE65 was injected subretinally into one eye of rd12 and Rpe65(-/-)::Rho(-/-) mice at postnatal day 14 (P14). Contralateral rd12 eyes were injected later, at P35. Rd12 behavioral testing revealed that rod vision loss was prevented with either P14 or P35 treatment, whereas cone vision was only detected after P14 treatment. Consistent with this observation, P35 treatment only restored rod electroretinogram (ERG) signals, a result likely due to reduced cone densities at this time point. For Rpe65(-/-)::Rho(-/-) mice in which there is no confounding rod contribution to the ERG signal, cone cells and cone-mediated ERGs were also maintained with treatment at P14. This work establishes that a self-complimentary AAV5 vector can restore substantial visual function in two genetically distinct models of Rpe65 deficiency within 4 days of treatment. In addition, this therapy prevents cone degeneration but only if administered before extensive cone degeneration, thus supporting continuation of current Leber's congenital amaurosis-2 clinical trials with an added emphasis on cone subtype analysis and early intervention.

Local correlation of expression profiles with gene annotations--proof of concept for a general conciliatory method.

MOTIVATION: Integrated analysis of expression data and gene ontology annotations is a prime example of biological data that need co-explanatory interpretation. This particular application is used to validate a new method for integrated analysis of varied biological information. RESULTS: The proposed method consists of determining local correlation coefficients and the corresponding P-values calculated per biological entity. This measure considers the combined intensity and significance of the agreement or disagreement, between two data sources about the same biological entity. The method is applied to the integrated analysis of gene expression and annotation of two gene sets, one from yeast and other from mouse. The potential of the method to generate accurate mechanistic hypothesis is also demonstrated. Specially, negative correlation results pose a new kind of biological hypothesis. Method performance was compared with annotation enrichment methods, and optimal conditions for the superiority of local correlation results are discussed.

Plasminogen kringle 5 reduces vascular leakage in the retina in rat models of oxygen-induced retinopathy and diabetes.

AIMS/HYPOTHESIS: Retinal vascular leakage is an early pathological feature in diabetic retinopathy and can lead to macular oedema and loss of vision. Previously we have shown that plasminogen kringle 5 (K5), an angiogenic inhibitor, inhibits retinal neovascularisation in the rat model of oxygen-induced retinopathy (OIR). The purpose of this study was to examine the effect of K5 on vascular leakage in the retina. METHODS: Neonatal rats were exposed to hyperoxia to induce OIR. Diabetes was induced in adult rats by injecting streptozotocin. Vascular permeability was measured by Evans blue method. Expression of vascular endothelial growth factor (VEGF) was evaluated using immunohistochemistry and western blot analysis. RESULTS: Rats with OIR and diabetes showed abnormal vascular hyperpermeability in the retina and iris. Intravitreal injection of K5, reduced vascular permeability in both animal models, but did not affect permeability in normal rats. K5 reduced vascular permeability at doses substantially lower than that required for inhibition of retinal neovascularisation. The K5-induced reduction in vascular permeability correlated with its down-regulation of VEGF expression in the retina. Moreover, K5 inhibited IGF-1-induced hyperpermeability, which is known to arise through up-regulation of endogenous VEGF expression. However, K5 had no effect on the hyperpermeability induced by injection of exogenous VEGF. CONCLUSIONS/INTERPRETATION: Very low doses of K5 reduce pathological vascular leakage in the retina. K5 thus has therapeutic potential in the treatment of diabetic macular oedema. This effect can be ascribed, at least in part, to the down-regulation of endogenous VEGF expression.

The noradrenergic system of aged GDNF heterozygous mice.

Glial cell line-derived neurotrophic factor (GDNF) is a trophic factor for noradrenergic (NE) neurons of the pontine nucleus locus coeruleus (LC). Decreased function of the LC-NE neurons has been found during normal aging and in neurodegenerative disorders. We have previously shown that GDNF participates in the differentiation of LC-NE neurons during development. However, the continued role of GDNF for LC-NE neurons during maturation and aging has not been addressed. We examined alterations in aged mice that were heterozygous for the GDNF gene (Gdnf+/-). Wild-type (Gdnf+/+) and Gdnf+/- mice (18 months old) were tested for locomotor activity and brain tissues were collected for measuring norepinephrine levels and uptake, as well as for morphological analysis. Spontaneous locomotion was reduced in Gdnf+/- mice in comparison with Gdnf+/+ mice. The reduced locomotor activity of Gdnf+/- mice was accompanied by reductions in NE transporter activity in the cerebellum and brain stem as well as decreased norepinephrine tissue levels in the LC. Tyrosine hydroxylase (TH) immunostaining demonstrated morphological alterations of LC-NE cell bodies and abnormal TH-positive fibers in the hippocampus, cerebellum, and frontal cortex of Gdnf+/- mice. These findings suggest that the LC-NE system of Gdnf+/- mice is impaired and suggest that GDNF plays an important role in continued maintenance of this neuronal system throughout life.

Lack of p75 receptor does not protect photoreceptors from light-induced cell death.

Rod photoreceptors are susceptible to light-induced cell death. Previous results have suggested that the neurotrophin receptor p75 in Müller cells controls photoreceptor cell death during light-exposure by suppressing trophic factor release; and consequently, if p75 is blocked or eliminated during light-exposure, apoptosis is delayed. We explored this question by examining photoreceptor cell survival in albino p75(-/-) mice as well as their heterozygous and homozygous littermates. Photoreceptor cell death was examined in semi-thin sections by counting the remaining rows of photoreceptors. No difference in the amount of cell death was found between p75(+/+) and p75(-/-) animals, whereas the single copy of p75 in the heterozygous p75(+/-) mice provided significant neuroprotection. Cell death in the wild-type animals may indeed be mediated by p75, whereas other known apoptosis pathways may be activated in the p75(-/-) mice. The pro-apoptotic activity of the p75 receptor may have been partially suppressed in the heterozygous p75(+/-) mice by the silencing effect of the Trk receptor. Thus, our results suggest that p75 signaling does not mediate the main apoptosis pathway activated during light-damage.

The Noradrenergic System of Aged GDNF Heterozygous Mice.

Glial cell line-derived neurotrophic factor (GDNF) is a trophic factor for noradrenergic (NE) neurons of the pontine nucleus locus coeruleus (LC). Decreased function of the LC-NE neurons has been found during normal aging and in neurodegenerative disorders. We have previously shown that GDNF participates in the differentiation of LC-NE neurons during development. However, the continued role of GDNF for LC-NE neurons during maturation and aging has not been addressed. We examined alterations in aged mice that were heterozygous for the GDNF gene (Gdnf+/-). Wild-type (Gdnf+/+) and Gdnf+/- mice (18 months old) were tested for locomotor activity and brain tissues were collected for measuring norepinephrine levels and uptake, as well as for morphological analysis. Spontaneous locomotion was reduced in Gdnf+/- mice in comparison with Gdnf+/+ mice. The reduced locomotor activity of Gdnf +/- mice was accompanied by reductions in NE transporter activity in the cerebellum and brain stem as well as decreased norepinephrine tissue levels in the LC. Tyrosine hydroxylase (TH) immunostaining demonstrated morphological alterations of LC-NE cell bodies and abnormal TH-positive fibers in the hippocampus, cerebellum, and frontal cortex of Gdnf+/- mice. These findings suggest that the LC-NE system of Gdnf+/- mice is impaired and suggest that GDNF plays an important role in continued maintenance of this neuronal system throughout life.

Robot-aided neurorehabilitation: from evidence-based to science-based rehabilitation.

There is no "magic bullet" in rehabilitation. In the absence of direct neural transplants, neurological rehabilitation is an arduous process. We have pioneered the clinical application of robotics in stroke rehabilitation and have shown evidence of the positive impact of targeted exercise on stroke recovery. In this article, we will review results obtained in the initial clinical trials with 96 stroke patients at the Burke Rehabilitation Hospital. We will provide evidence that robot-aided training enhances recovery, that this enhanced recovery is sustained in the long term, and that this recovery is not due to a general physiological improvement--in fact, it appears to be limb and muscle group specific. An evidence-based approach must now segue into a more scientific approach to stroke rehabilitation. Given the length of the required protocols and patients' variability and limited census, the practical limitations of the evidence-based approach are self-evident and extend trials for years. Each patient and lesion is unique in stroke rehabilitation, so there is no reason to believe that a "one-size-fits-all" optimal treatment exists. To optimize therapy for individual patients, we need science-based models. In this article, we will summarize the scientific tools and models that we are investigating and present some of the results to date.

Neurotrophin receptor TrkB activation is not required for the postnatal survival of retinal ganglion cells in vivo.

During early postnatal development, apoptosis of retinal ganglion cells (RGCs) is regulated by target contact with the optic tectum. The neurotrophins BDNF and NT-4, but not NGF, prevent the apoptosis of retinal ganglion cells that is otherwise observed after target ablation or axotomy. Thus receptors activated by BDNF and NT-4 are candidates to mediate the early postnatal survival of RGCs. BDNF and NT-4, but not NGF, bind to all isoforms of the receptor TrkB, whether or not they contain a tyrosine kinase domain. To examine the roles of TrkB receptor isoforms in early postnatal survival, we compared RGC numbers in wild-type mice to those in a mutant lacking all isoforms of TrkB. Surprisingly, no reduction in RGCs was observed in the mutant at postnatal day 16, the latest age at which these animals are consistently viable, so TrkB signaling is not essential for target-dependent survival of these cells. In wild-type mice, RGCs also are lost gradually during adulthood, possibly due to oxidative stress. To determine whether TrkB signaling regulates this phase of RGC degeneration, RGC numbers were examined in a viable mutant of TrkB that expresses only about 25% the normal level of TrkB receptor kinase. Compared to controls, approximately 20% of the RGC were lost in mutant 3-month-old-animals. Thus, TrkB signaling is not required for survival of RGCs during the period of target-dependent survival, but does appear to reduce degeneration of RGCs in adult animals.

Evaluation of a simple method for minimizing iatrogenic blood loss from discard volumes in critically ill newborns and children.

OBJECTIVE: To validate a simple method avoiding discard volumes in pediatric patients with indwelling arterial and venous lines. DESIGN: Zero-discarding was achieved by passive extracorporeal arteriovenous backflow via standard single pressure transducer equipment. We tested backflow distances (10, 20 and 30 cm beyond the sampling port), corresponding to withdrawal volumes of 0.6 ml, 0.8 ml and 1.0 ml, respectively, in comparison to conventional sampling with discard of 0.6 ml. With the backflow technique, the "withdrawal volume" was flushed back to the patient after sampling. We enrolled 120 patients who were allocated to either of the following paired sampling procedures: 10 cm versus conventional, 20 cm versus conventional, 30 cm versus conventional and two paired conventional samples. The order of the sampling was randomly allocated. Bias and precision were determined using Bland-Altman diagrams and algorithms. RESULTS: No appreciable difference was found for blood gases, hemoglobin, potassium and calcium between the backflow technique and conventional sampling. Sodium results and blood glucose showed a bias towards higher values with the backflow technique (mean difference, sodium, 0.9 mmol/l; glucose, mean difference 0.5 mmol/l, standard deviation 0.44 mmol/l). CONCLUSIONS: The backflow technique provides reliable results for blood gases and electrolytes. However, in patients at risk of hypoglycemia, the backflow method should not be used to monitor blood glucose levels.

Intensity modulated proton therapy: a clinical example.

In this paper, we report on the clinical application of fully automated three-dimensional intensity modulated proton therapy, as applied to a 34-year-old patient presenting with a thoracic chordoma. Due to the anatomically challenging position of the lesion, a three-field technique was adopted in which fields incident through the lungs and heart, as well as beams directed directly at the spinal cord, could be avoided. A homogeneous target dose and sparing of the spinal cord was achieved through field patching and computer optimization of the 3D fluence of each field. Sensitivity of the resultant plan to delivery and calculational errors was determined through both the assessment of the potential effects of range and patient setup errors, and by the application of Monte Carlo dose calculation methods. Ionization chamber profile measurements and 2D dosimetry using a scintillator/CCD camera arrangement were performed to verify the calculated fields in water. Modeling of a 10% overshoot of proton range showed that the maximum dose to the spinal cord remained unchanged, but setup error analysis showed that dose homogeneity in the target volume could be sensitive to offsets in the AP direction. No significant difference between the MC and analytic dose calculations was found and the measured dosimetry for all fields was accurate to 3% for all measured points. Over the course of the treatment, a setup accuracy of +/-4 mm (2 s.d.) could be achieved, with a mean offset in the AP direction of 0.1 mm. Inhalation/exhalation CT scans indicated that organ motion in the region of the target volume was negligible. We conclude that 3D IMPT plans can be applied clinically and safely without modification to our existing delivery system. However, analysis of the calculated intensity matrices should be performed to assess the practicality, or otherwise, of the plan.

Gene dosage effect of the TrkB receptor on rod physiology and biochemistry in juvenile mouse retina.

PURPOSE: To strengthen our understanding about the role of trkB in rod development by correlating functional and biochemical retinal phenotypes with levels of trkB expression in two independently created trkB transgenic lines. METHODS: Juvenile mice that carried two hypomorphic trkB alleles (trkBfbz/fbz) expressing roughly 25% of normal trkB, and their heterozygous (trkB+/fbz) and wild type (WT) littermates were tested with electroretinographic (ERG) protocols that isolate rod driven responses. Rod development was assessed histologically (outer segment length) and spectrophotometrically (rhodopsin content). Functional and biochemical data were compared to those obtained from mice that have trkB removed completely (trkB-/-). RESULTS: (1) Retinal rod function and morphology was unaffected by a loss of up to 50% of trkB. (2) However, reducing trkB below a critical threshold (<50%) significantly reduced rhodopsin content and outer segment length, resulting in reduced a- and b-wave amplitudes and slower kinetics. (3) A second threshold was determined for rod to bipolar cell signaling, which requires the presence of at least 25% of wild type trkB levels. CONCLUSIONS: (1) These results demonstrated that rod biochemistry, physiology and synaptic signaling are compromised in a gene dosage dependent manner when the expression of trkB is reduced in transgenic mice. (2) This study confirmed our previous conclusion that the knockout of trkB expression altered rod development, because this gene product is essential for normal rod maturation and not because of alternative indirect mechanisms. (3) More generally, this study showed that the specificity of complex phenotypes can be investigated in gene knockout mice, if a gene dosage study is performed.

Role of neurotrophin receptor TrkB in the maturation of rod photoreceptors and establishment of synaptic transmission to the inner retina.

Brain-derived neurotrophic factor (BDNF) acts through TrkB, a receptor with kinase activity, and mitigates light-induced apoptosis in adult mouse rod photoreceptors. To determine whether TrkB signaling is necessary for rod development and function, we examined the retinas of mice lacking all isoforms of the TrkB receptor. Rod migration and differentiation occur in the mutant retina, but proceed at slower rates than in wild-type mice. In postnatal day 16 (P16) mutants, rod outer segment dimensions and rhodopsin content are comparable with those of photoreceptors in P12 wild type (WT). Quantitative analyses of the photoreceptor component in the electroretinogram (ERG) indicate that the gain and kinetics of the rod phototransduction signal in dark-adapted P16 mutant and P12 WT retinas are similar. In contrast to P12 WT, however, the ERG in mutant mice entirely lacks a b-wave, indicating a failure of signal transmission in the retinal rod pathway. In the inner retina of mutant mice, although cells appear anatomically and immunohistochemically normal, they fail to respond to prolonged stroboscopic illumination with the normal expression of c-fos. Absence of the b-wave and failure of c-fos expression, in view of anatomically normal inner retinal cells, suggest that lack of TrkB signaling causes a defect in synaptic signaling between rods and inner retinal cells. Retinal pigment epithelial cells and cells in the inner retina, including Müller, amacrine, and retinal ganglion cells, express the TrkB receptor, but rod photoreceptors do not. Moreover, inner retinal cells respond to exogenous BDNF with c-fos expression and extracellular signal-regulated kinase phosphorylation. Thus, interactions of rods with TrkB-expressing cells must be required for normal rod development.

Glucagon-like peptide-1 promotes satiety and reduces food intake in patients with diabetes mellitus type 2.

Glucagon-like peptide-1-(7-36) amide (GLP-1) is an incretin hormone of the enteroinsular axis. Recent experimental evidence in animals and healthy subjects suggests that GLP-1 has a role in controlling appetite and energy intake in humans. We have therefore examined in a double-blind, placebo-controlled, crossover study in 12 patients with diabetes type 2 the effect of intravenously infused GLP-1 on appetite sensations and energy intake. On 2 days, either saline or GLP-1 (1.5 pmol. kg-1. min-1) was given throughout the experiment. Visual analog scales were used to assess appetite sensations; furthermore, food and fluid intake of a test meal were recorded, and blood was sampled for analysis of plasma glucose and hormone levels. GLP-1 infusion enhanced satiety and fullness compared with placebo (P = 0.028 for fullness and P = 0.026 for hunger feelings). Energy intake was reduced by 27% by GLP-1 (P = 0.034) compared with saline. The results demonstrate a marked effect of GLP-1 on appetite by showing enhanced satiety and reduced energy intake in patients with diabetes type 2.

Endogenous opiates in the chick retina and their role in form-deprivation myopia.

In this study, the possible role of the retinal enkephalin system in form-deprivation myopia (FDM) in the chick eye was investigated. Daily intravitreal injection of the nonspecific opiate antagonist naloxone blocked development of FDM in a dose-dependent manner, while injection of the opiate agonist morphine had no effect at any dose tested. The ED50 for naloxone (calculated maximum concentration in the vitreous) was found to be in the low picomolar range. The results using receptor-subtype-specific drugs were contradictory. Drugs specific for mu and delta receptors had no effect on FDM. The kappa-specific antagonist nor-binaltorphimine (nor-BNI) reduced FDM by about 50% at maximum daily retinal doses ranging between 4 x 10(-10) and 4 x 10(-7) M, while the kappa-specific agonist U50488 blocked FDM in a dose-dependent manner with an ED50 between 5 x 10(-8) and 5 x 10(-7) M. Met-enkephalin immunoreactivity (ME-IR) was localized immunocytochemically to a subset of amacrine cells (ENSLI cells) and their neurites in the inner plexiform layer (IPL). As reported previously, ENSLI cells from untreated chick retinas showed a cyclical pattern of immunoreactivity, with increased immunoreactivity in the light compared to the dark. Form-deprivation did not appear to change this pattern. Amounts of preproenkephalin mRNA from normal or form-deprived eyes were approximately the same under all conditions. Daily injection of naloxone, however, did increase ME-IR in the dark. These results suggest that naloxone may affect release of enkephalin from the ENSLI cells. The results as presented are inconclusive with regards to the role of the enkephalin system in FDM. While the kappa receptor may participate, there is no conclusive evidence here for a direct effect of opiate receptors. The effect of naloxone on form-deprived eyes may be due to its effect on release of peptides from the ENSLI cells.

Basic fibroblast growth factor, its high- and low-affinity receptors, and their relationship to form-deprivation myopia in the chick.

Form deprivation myopia in chickens is a widely accepted model to study visually-regulated postnatal ocular growth. Recently we showed that basic fibroblast growth factor-2 provides a "stop" signal for the growing eye. To understand further its action, we have localized basic fibroblast growth factor-2 and its low- and high-affinity receptors in the chicken eye, and determined the localization of basic fibroblast growth factor receptors in the inner plexiform layer with respect to that of neurotransmitter systems known to play a role in form-deprivation myopia. By immunocytochemistry and in situ hybridization, two complementary methods, we found that nearly all cells in the retina, and scleral chondrocytes, contain basic fibroblast growth factor-2 protein and messenger RNA as well as high-affinity basic fibroblast growth factor receptor protein and messenger RNA. Immunocytochemical localization of basic fibroblast growth factor-2 binding sites (a high resolution alternative to autoradiography), combined with N-glycanase and heparitinase treatment or heparin competition, revealed additional binding sites in specific synaptic layers of the inner plexiform layer and low-affinity binding sites in the choroid and optic fibre layer. Some binding sites in the synaptic layers were found to co-stratify with neurites of dopamine-, vasoactive intestinal polypeptide- or enkephalin-containing amacrine cells, suggesting that basic fibroblast growth factor-2 could modulate synaptic transmission to or from these cells. Form deprivation did not affect the levels of basic fibroblast growth factor receptor-1 messenger RNA in retina/retinal pigment epithelium/choroid (Northern blotting), but it abolished the decrease in amount of extractable basic fibroblast growth factor normally observed in the dark (Western blotting). The results are discussed with respect to previous findings on basic fibroblast growth factor-2 and basic fibroblast growth factor receptor-1 localization in the avian and other vertebrate eyes, and their relevance to form-deprivation myopia. The widespread distribution of basic fibroblast growth factor-2 and its receptor makes it impossible to predict which cells might mediate the action of basic fibroblast growth factor-2 in form-deprivation myopia. However, the alteration in amounts of extractable retinal basic fibroblast growth factor-2 in form-deprived, dark-adapted retinas, in which basic fibroblast growth factor-2 probably serves as a "stop" signal for ocular growth, is consistent with a role for basic fibroblast growth factor-2 in the regulation of ocular growth.

Localization of putative dopamine D2-like receptors in the chick retina, using in situ hybridization and immunocytochemistry.

The actions of dopamine are mediated by 5 or more receptor subtypes, any of which may be coupled by G-proteins to adenylate cyclase (D1-family: stimulatory, D2-family: inhibitory or no action). Postnatal ocular growth in the chick is a vision-dependent mechanism which involves D2-type receptors in either the retina or the retinal pigment epithelium (RPE). Although the dopaminergic amacrine cells are well described in the chick retina, only D2-receptors, but not D3- and D4-receptors have been clearly localized, and the cells that express them have not been identified. In this study we showed that immunoreactive D2/3-receptor protein is localized to the photoreceptor inner segments, outer and inner plexiform layer and ganglion cell layer, as described previously (Wagner et al., J. Comp. Neurol., 330 (1993) 1-13). D2-receptor mRNA was localized to cell bodies in all nuclear layers of the retina, whereas D4-receptor mRNA was restricted to the inner half of the retina. Immunoreactive D2-type receptors and their mRNA were observed also in the basal region of the RPE. Because of the widespread distribution of both D2- and D4-receptor mRNA in the chick retina and RPE and the lack of D3- and D4-receptor-specific antibodies, we were unable to identify which of the D2/3/4-receptor-bearing cells are involved in controlling ocular growth.

Stimulation of dopaminergic amacrine cells by stroboscopic illumination or fibroblast growth factor (bFGF, FGF-2) injections: possible roles in prevention of form-deprivation myopia in the chick.

Form-deprivation myopia (FDM) in the chick is a popular model for studying the postnatal regulation of ocular growth. Using this model, we have shown previously that dopamine and FGF-2 can counteract the effects of form-deprivation, thereby producing emmetropia. In the present study, we tested the hypothesis that the emmetropizing effects of flickering light and intraocular injections of FGF-2 in the chick are mediated by the activity of dopaminergic retinal amacrine cells. We have assessed the rate of dopamine synthesis in the retina by measuring the accumulation of 3,4-dihydroxyphenylalanine (DOPA). We found that form-deprivation reduces the rate of dopamine synthesis in the light-adapted retina, and that the normal rate of dopamine synthesis in the light can be restored by stroboscopic illumination at frequencies around 10 Hz. By labeling cells immunocytochemically we have shown that the synthesis of c-fos, a putative transcriptional regulator of the tyrosine hydroxylase gene, is induced in dopaminergic amacrine cells by stroboscopic illumination at around 10 Hz. These observations are consistent with a critical role for dopaminergic amacrine cells in the regulation of ocular growth by intermittent illumination. We have found also that intraocular injections of FGF-2 cause emmetropization without altering levels of expression of c-fos, amounts of tyrosine hydroxylase, or rates of dopamine synthesis with respect to vehicle-injected controls. We conclude that FGF acts either in parallel to or downstream from the dopaminergic amacrine cells, rather than through them. We observed that intravitreal injection per se induces high levels of c-fos expression in both form-deprived and non-deprived retinas, and causes partial emmetropization in form-deprived eyes, while inhibiting dopamine synthesis in non-deprived retinas. It is likely, therefore, that injection stimulates the production and/or release of unknown factors whose diverse effects on ocular growth and dopamine metabolism are mediated by complex pathways. Taken together, our results are consistent with the view that the retinal circuitry that controls postnatal ocular growth in the chick involves multiple messengers and pathways.

Long-term platinum excretion in patients treated with cisplatin.

The purpose of this study was to determine long-term renal platinum excretion after chemotherapy with cisplatin. We examined urinary platinum concentrations in 23 men at 150-3022 days after anticancer treatment for testicular neoplasm. Spot urine samples were analyzed by voltammetry. This new, subtle method with a detection limit of 2 pg platinum allows determination of even the natural background level. Urinary platinum concentrations in our patients ranged between 0.74 and 77.24 micrograms/g creatinine, depending on the total delivered dose and follow-up period. Regression analysis of the data showed two phases of long-term renal platinum excretion, one occurring at between 150 and 900 days of follow-up and the other with an onset at 900 days after cisplatin administration (r1(2) = 0.82, r2(2) = 0.88). Two biological half-lives of 160 and 720 days were calculated. Our results show that urinary platinum concentrations determined at 8 years after cisplatin therapy are 40 times higher than the background level (up to 0.02 micrograms/g creatinine). Our findings on the long-term pharmacokinetics of this anticancer agent may facilitate further studies on sites of platinum storage in the human body as well as clinical studies on the late adverse effects of cisplatin.

Gastrointestinal intramural hematoma, a complication of endoscopic injection methods for bleeding peptic ulcers: a case series.

In a prospective study, all patients with peptic ulcer bleeding were documented between February 1984 and April 1992. A total of 227 patients were treated by local injection of epinephrine followed by laser application and injection of polidocanol or fibrin tissue adhesive. In five of these patients, intramural hematomas developing at the former bleeding site one to three days after endoscopic treatment were observed. The presenting symptoms were abdominal pain, nausea, and vomiting. The diagnosis was established by endoscopy, abdominal ultrasound, computed tomography, or laparotomy. In four of our five patients, the bleeding site and hematoma were located in the duodenum. All patients suffered from severe underlying diseases, and showed a clear disturbance of coagulation parameters. In three patients, acute pancreatitis occurred concurrently with the hematoma, probably due to obstruction of the papilla of Vater or compression of the pancreas caused by the hematoma.