Circadian and Dopaminergic Regulation of Fatty Acid Oxidation Pathway Genes in Retina and Photoreceptor Cells.

The energy metabolism of the retina might comply with daily changes in energy demand and is impaired in diabetic retinopathy-one of the most common causes of blindness in Europe and the USA. The aim of this study was to investigate putative adaptation of energy metabolism in healthy and diabetic retina. Hence expression analysis of metabolic pathway genes was performed using quantitative polymerase chain reaction, semi-quantitative western blot and immunohistochemistry. Transcriptional profiling of key enzymes of energy metabolism identified transcripts of mitochondrial fatty acid ß-oxidation enzymes, i.e. carnitine palmitoyltransferase-1α (Cpt-1α) and medium chain acyl-CoA dehydrogenase (Acadm) to display daily rhythms with peak values during daytime in preparations of the whole retina and microdissected photoreceptors. The cycling of both enzymes persisted in constant darkness, was dampened in mice deficient for dopamine D4 (D4) receptors and was altered in db/db mice-a model of diabetic retinopathy. The data of the present study are consistent with circadian clock-dependent and dopaminergic regulation of fatty acid oxidation in retina and its putative disturbance in diabetic retina.

Pgc-1α and Nr4a1 Are Target Genes of Circadian Melatonin and Dopamine Release in Murine Retina.

PURPOSE: The neurohormones melatonin and dopamine mediate clock-dependent/circadian regulation of inner retinal neurons and photoreceptor cells and in this way promote their functional adaptation to time of day and their survival. To fulfill this function they act on melatonin receptor type 1 (MT1 receptors) and dopamine D4 receptors (D4 receptors), respectively. The aim of the present study was to screen transcriptional regulators important for retinal physiology and/or pathology (Dbp, Egr-1, Fos, Nr1d1, Nr2e3, Nr4a1, Pgc-1α, Rorß) for circadian regulation and dependence on melatonin signaling/MT1 receptors or dopamine signaling/D4 receptors. METHODS: This was done by gene profiling using quantitative polymerase chain reaction in mice deficient in MT1 or D4 receptors. RESULTS: The data obtained determined Pgc-1α and Nr4a1 as transcriptional targets of circadian melatonin and dopamine signaling, respectively. CONCLUSIONS: The results suggest that Pgc-1α and Nr4a1 represent candidate genes for linking circadian neurohormone release with functional adaptation and healthiness of retina and photoreceptor cells.

Transcriptional regulation of nucleoredoxin-like genes takes place on a daily basis in the retina and pineal gland of rats.

The nucleoredoxin-like gene Nxnl1 (Txnl6) and its paralogue Nxnl2 encode the rod-derived cone viability factors (RdCVF and RdCVF2), which increase the resistance to photooxidative damage and have therapeutic potential for the survival of cones in retinitis pigmentosa. In this study, the transcription of Nxnl genes was investigated as a function of the day/night cycle in rats. The transcript levels of Nxnl1 and Nxnl2 were seen to display daily rhythms with steadily increasing values during the light phase and peak expression around dark onset in preparations of whole retina, photoreceptor cells and-but only in regard to Nxnl1-in photoreceptor-related pinealocytes. The cycling of Nxnl1 but not that of Nxnl2 persisted in constant darkness in the retina. This suggests that daily regulation of Nxnl1 is driven by a circadian clock, whereas that of Nxnl2 is promoted by environmental light. The present data indicate clock- and light-dependent regulations of nucleoredoxin-like genes that may be part of a protective shield against photooxidative damage.

Photoreceptor cells display a daily rhythm in the orphan receptor Esrrß.

PURPOSE: Nuclear orphan receptors are critical for the development and long-term survival of photoreceptor cells. In the present study, the expression of the nuclear orphan receptor Esrrß--a transcriptional regulator of energy metabolism that protects rod photoreceptors from dystrophy--was tested under daily regulation in the retina and photoreceptor cells. METHODS: The daily transcript and protein amount profiles were recorded in preparations of the whole retina and microdissected photoreceptor cells using quantitative PCR (qPCR) and western blot analysis. RESULTS: Esrrß displayed a daily rhythm with elevated values at night in the whole retina and enriched photoreceptor cells. Daily regulation of Esrrß mRNA depended on light input but not on melatonin, and evoked a corresponding rhythm in the Esrrß protein. CONCLUSIONS: The data presented in this study indicate that daily regulation of Esrrß in photoreceptor cells may contribute to their adaptation to 24-h changes in metabolic demands.

Transcriptional analysis of rat photoreceptor cells reveals daily regulation of genes important for visual signaling and light damage susceptibility.

Photoreceptor cells face the challenge of adjusting their function and, possibly, their susceptibility to light damage to the marked daily changes in ambient light intensity. To achieve a better understanding of photoreceptor adaptation at the transcriptional level, this study aimed to identify genes which are under daily regulation in photoreceptor cells using microarray analysis and quantitative PCR. Included in the gene set obtained were a number of genes which up until now have not been shown to be expressed in photoreceptor cells, such as Atf3 (activating transcription factor 3) and Pde8a (phosphodiesterase 8A), and others with a known impact on phototransduction and/or photoreceptor survival, such as Grk1 (G protein-coupled receptor kinase 1) and Pgc-1α (peroxisome proliferator-activated receptor γ, coactivator 1alpha). According to their daily dynamics, the genes identified could be clustered in two groups: those with peak expression during the second part of the day which are uniformly promoted to cycle by light/dark transitions and those with peak expression during the second part of the night which are predominantly driven by a clock. Since Grk1 and Pgc-1α belong in the first group, the present results support a concept in which transcriptional regulation of genes by ambient light contributes to the functional adjustment of photoreceptor cells over the 24-h period.

The retinal clock drives the expression of Kcnv2, a channel essential for visual function and cone survival.

PURPOSE: The gene Kcnv2 codes for the voltage-gated potassium channel subunit Kv8.2, which can coassemble with Kv2.1 subfamily members to constitute functional voltage-gated potassium channels. Mutations in the Kcnv2 gene result in a retinal disorder designated "cone dystrophy with supernormal rod response (CDSRR)," revealing that Kcnv2 is essential for visual processing and cone survival. The aim of this study was to determine whether expression of Kcnv2 and Kv2.1 is under circadian regulation and may thus contribute to the clock-driven adjustment of photoreceptor function. METHODS: Expression of the genes was recorded in preparations of the whole retina and microdissected retinal neurons by using quantitative polymerase chain reaction and Western blot. RESULTS: The transcript levels of Kcnv2 and Kv2.1 in preparations of whole retina and photoreceptor cells were found to display daily rhythms, with elevated values during the night. For Kcnv2 this rhythm was shown to evoke a corresponding rhythm in Kv8.2, the protein product of this gene. The daily changes in retinal Kcnv2 and Kv2.1 mRNA levels persisted under constant darkness and are therefore driven by the endogenous retinal clock system, which itself is entrained by light. CONCLUSIONS: The present data provide evidence that the transcriptional regulation of Kcnv2 and Kv2.1 is a way through which the retinal clock system drives the functional adaptation of visual function to the marked daily changes in environmental lighting conditions.

Impact of single-dose application of TGF-ß, copper peptide, stanozolol and ascorbic acid in hydrogel on midline laparatomy wound healing in a diabetic mouse model.

Despite numerous advances and improvements in surgical techniques the incidence of incisional hernias after laparotomy remains high. The aim of this study was to investigate possible effects of single application of ascorbic acid, stanozolol, a synthetic anabolic steroid, copper peptide and transforming growth factor-ß (TGF-ß) on laparotomy wound healing in an incisional wound model in diabetic mice. After diabetes induction with streptozotozin in Balb-c mice, midline laparatomies were carried out. Closure of the linea alba was followed by single-dose application of the agents dissolved in a hydrogel before skin closure. The functional outcome was assessed in terms of maximum tensile strength. In addition, vessel densities, collagen contents and proliferation, were measured. The breaking strength of the skin 14 days after surgery was significantly higher in ascorbic acid (ΑΑ)-treated incisional wounds, whereas the other agents did not show a significantly better functional outcome. No significant differences were seen in vessel densities. Collagen type III contents was higher in the ΑΑ-treated animals, whereas the percentage of Ki67-positive nuclei was lower compared to the other groups. These data underline the positive effect of topically applied ascorbic acid in wound healing.

Priming with a combination of proangiogenic growth factors enhances wound healing in streptozotocin-induced diabetes in mice.

BACKGROUND: Numerous proangiogenic growth factors have been shown to improve impaired wound healing. This study evaluated the effects of subcutaneous pretreatment with a combination of proangiogenic growth factors on wound closure, mechanical properties, vessel density, and morphology. METHODS: Thirty-six Balb/c mice with streptozotocin-induced diabetes were divided into 3 groups. A mixture of VEGF (35.0 µg), bFGF (2.5 µg), and PDGF (3.5 µg) was administered subcutaneously 3, 5, and 7 days prior to wounding in the first group, whereas the second group received three doses of 3.5 µg PDGF. Wound sizes were assessed daily and the repaired tissues were harvested 7 days after wound closure. RESULTS: Complete closure (≥95% healing of initial wound area) was reached in all proangiogenic pretreated animals by day 17, whereas the PDGF monotherapy group needed up to 20 days for complete closure. By the time of tissue harvesting on day 24, complete closure was not reached in all control animals. Punch biopsy material revealed 1.6-fold higher vessel densities in the proangiogenic combination-pretreated group than in the controls. CONCLUSIONS: Proangiogenic priming revealed several significant effects on diabetic wound healing: faster time to closure, a higher vessel density, and improved functional outcome.

Phosphodiesterase 10A in the rat pineal gland: localization, daily and seasonal regulation of expression and influence on signal transduction.

The cyclic nucleotide phosphodiesterase 10A (PDE10A) is highly expressed in striatal spiny projection neurons and represents a therapeutic target for the treatment of psychotic symptoms. As reported previously [J Biol Chem 2009; 284:7606-7622], in this study PDE10A was seen to be additionally expressed in the pineal gland where the levels of PDE10A transcript display daily changes. As with the transcript, the amount of PDE10A protein was found to be under daily and seasonal regulation. The observed cyclicity in the amount of PDE10A mRNA persists under constant darkness, is blocked by constant light and is modulated by the lighting regime. It therefore appears to be driven by the master clock in the suprachiasmatic nucleus (SCN). Since adrenergic agonists and dibutyryl-cAMP induce PDE10A mRNA, the in vitro clock-dependent control of Pde10a appears to be mediated via a norepinephrine → ß-adrenoceptor → cAMP/protein kinase A signaling pathway. With regard to the physiological role of PDE10A in the pineal gland, the specific PDE10A inhibitor papaverine was seen to enhance the adrenergic stimulation of the second messenger cAMP and cGMP. This indicates that PDE10A downregulates adrenergic cAMP and cGMP signaling by decreasing the half-life of both nucleotides. Consistent with its effect on cAMP, PDE10A inhibition also amplifies adrenergic induction of the cAMP-inducible gene arylalkylamine N-acetyltransferase (Aanat) which codes the rate-limiting enzyme in pineal melatonin formation. The findings of this study suggest that Pde10a expression is under circadian and seasonal regulation and plays a modulatory role in pineal signal transduction and gene expression.

Priming with a combination of proangiogenic growth factors improves wound healing in normoglycemic mice.

Growth factors and/or angiogenic factors are supposed to improve wound healing. The aim of our study was to evaluate the effects of subcutaneous pretreatment with combinatory proangiogenic factors on wound closure, mechanical properties, vessel density and morphology. Twenty-eight Balb/c mice were divided equally into two groups. A mixture of VEGF (35.0 µg), bFGF (2.5 µg) and PDGF (3.5 µg) was administered subcutaneously 3, 5 and 7 days to 14 mice before full thickness skin punch biopsy wounding, whereas 14 control animals received three injections of 0.2 ml saline solution. Wound sizes were assessed daily and the repaired tissues were harvested 7 days after complete wound closure. Complete closure (≥ 95% healing of initial wound area) was reached in all proangiogenic pretreated animals on day 10, whereas controls needed 13 days for complete closure. Tensile strengths were nearly twofold higher compared to the controls (p ≤ 0.01). The punch biopsy material revealed 4.2-fold higher vessel densities in the proangiogenic pretreated group. On day 17, the vessel densities in the proangiogenic pretreated wounds were also 3.2-fold higher compared to the untreated controls. No significant differences were seen in the collagen ratio. Pretreatment with proangiogenic factors revealed several significant effects on wound healing: faster time to closure, a higher vessel density and a better functional outcome. These results suggest a beneficial effect of pretreatment with combinatory growth factors in mouse skin wounds without impaired wound healing. This might be exploited in further investigations in diabetic healing as a therapeutic approach for elective surgery.

Maximum forces acting on the abdominal wall: experimental validation of a theoretical modeling in a human cadaver study.

Incisional hernias following median laparotomy have a high incidence and recurrence rate after repair, so that a better understanding of the linea alba biomechanics is desirable. The mechanical stress exerted on the linea alba in living humans is primarily generated by the musculature. In this human cadaver study, intraabdominal pressure was simulated by insertion of a balloon that was increasingly filled to maximal pressures of 200 mbar. The related forces acting transversely on the linea alba at maximum pressure were found to be between 41.6 and 52.2N/cm (mean=45.9N/cm), which is in agreement with a recent modeling of the related forces.

Phosphodiesterase10A: abundance and circadian regulation in the retina and photoreceptor of the rat.

Phosphodiesterase10A (PDE10A) is a dual specific cyclic nucleotide phosphodiesterase that is specifically enriched in striatum and which has gained attention as a therapeutic target for psychiatric disorders. The present study shows that PDE10A is also highly expressed in retinal neurons including photoreceptors. The levels of PDE10A transcript and protein display daily rhythms which could be seen in preparations of the whole retina. Corresponding changes in PDE10A mRNA were seen in photoreceptors isolated using laser microdissection. This suggests that the expressional control of the photoreceptor Pde10a gene contributes to the observed cyclicity in the amount of retinal PDE10A. The daily rhythmicity in the retinal PDE10A mRNA amount is retained under constant darkness but can be blocked by constant light or modulated by the lighting regime. It therefore appears to be driven by the endogenous retinal clock system which itself is entrained by light. The findings presented place PDE10A in the context of the visual system and suggest a role of PDE10A in the adaptation of cyclic nucleotide signaling to daily changes in light intensity in retinal neurons including photoreceptors.

Unique clockwork in photoreceptor of rat.

In mammals, the retina contains a clock system that oscillates independently of the master clock in the suprachiasmatic nucleus and allows the retina to anticipate and to adapt to the sustained daily changes in ambient illumination. Using a combination of laser capture micro-dissection and quantitative PCR in the present study, the clockwork of mammalian photoreceptors has been recorded. The transcript amounts of the core clock genes Clock, Bmal1, Period1 (Per1), Per3, Cryptochrome2, and Casein kinase Iε in photoreceptors of rat retina have been found to undergo daily changes. Clock and Bmal1 peak with Per1 and Per3 around dark onset, whereas Casein kinase Iε and Cryptochrome2 peak at night. As shown for Clock, Per1, and Casein kinase Iε, the oscillation of transcript amounts results in daily changes of the protein products. The in-phase oscillation of Clock/Bmal1 with Pers and the rhythmic expression of Casein kinase Iε do not occur in molecular clocks of other tissues including the suprachiasmatic nucleus. Therefore, the findings presented suggest that the photoreceptor clock is unique not only in its position outside the clock hierarchy mastered by the suprachiasmatic nucleus, but also with regard to the intrinsic rhythmic properties of its molecular components.

Active zone proteins are dynamically associated with synaptic ribbons in rat pinealocytes.

Synaptic ribbons (SRs) are prominent organelles that are abundant in the ribbon synapses of sensory neurons where they represent a specialization of the cytomatrix at the active zone (CAZ). SRs occur not only in neurons, but also in neuroendocrine pinealocytes where their function is still obscure. In this study, we report that pinealocyte SRs are associated with CAZ proteins such as Bassoon, Piccolo, CtBP1, Munc13-1, and the motorprotein KIF3A and, therefore, consist of a protein complex that resembles the ribbon complex of retinal and other sensory ribbon synapses. The pinealocyte ribbon complex is biochemically dynamic. Its protein composition changes in favor of Bassoon, Piccolo, and Munc13-1 at night and in favor of KIF3A during the day, whereas CtBP1 is equally present during the night and day. The diurnal dynamics of the ribbon complex persist under constant darkness and decrease after stimulus deprivation of the pineal gland by constant light. Our findings indicate that neuroendocrine pinealocytes possess a protein complex that resembles the CAZ of ribbon synapses in sensory organs and whose dynamics are under circadian regulation.

How to optimize autonomic nerve preservation in total mesorectal excision: clinical topography and morphology of pelvic nerves and fasciae.

BACKGROUND: Urogenital dysfunction after rectal and pelvic surgery was significantly decreased with the introduction of nerve-preserving dissection and total mesorectal excision (TME). Profound topographic knowledge of the pelvic connective tissue spaces is indispensable for identification and preservation of autonomic pelvic nerves. The purpose of this cadaver study was to highlight the course of important autonomous nerve structures and to identify potential injury sites. METHODS: Eleven cadavers were dissected according to TME with subsequent preparation of the pelvic nerves. The pelves of further three cadavers were sliced horizontally and cubed. Specimens were harvested and processed for light microscopy and immunohistochemistry to analyze both fascia and the types of nerves and their localization. RESULTS: The neurovascular bundle, arising from the inferior pelvic plexus, shows the highest nerve density. At the lateral edge of Denonvilliers' fascia, it pierces the parietal pelvic fascia. Several fine nerve branches spread into the loose periprostatic tissue up to the prostate or pass the prostate toward the urinary bladder. En route, we consistently find perikarya of autonomic nerves. Within the mesorectum, nerve fibers are distributed heterogeneously with laterally high densities, ventrally and dorsally low densities. CONCLUSION: The highest risk for pelvic nerve damage-apart from lesions of the superior hypogastric plexus itself-is anterolaterally of the rectum where the neurovascular bundle releases from the pelvic sidewall. Careful dissection helps to identify and protect these nerve structures. The retroprostatic Denonvilliers' fascia contains no important nerve structures.

Murine microvideo endoscopy of the colonic microcirculation.

Natural orifice endoscopy in small animal models has been limited in the past by instrument size and optical performance. In this report, we investigate the feasibility of using a recently developed microvideo endoscopy system to evaluate the colon microcirculation. Using a murine model of acute colitis, microvideo endoscopy was useful in mapping the topography of inflammation as well as identifying relevant structures in the microcirculation. We conclude that natural orifice endoscopy is a useful method for the minimally invasive longitudinal assessment of the colonic mucosal microcirculation.

Resection of the mesopancreas (RMP): a new surgical classification of a known anatomical space.

BACKGROUND: Prognosis after surgical therapy for pancreatic cancer is poor and has been attributed to early lymph node involvement as well as to a strong tendency of cancer cells to infiltrate into the retropancreatic tissue and to spread along the peripancreatic neural plexuses. The objective of our study was to classify the anatomical-surgical layer of the mesopancreas and to describe the surgical principles relevant for resection of the mesopancreas (RMP). Immunohistochemical investigation of the mesopancreatic-perineural lymphogenic structures was carried out with the purpose of identifying possible routes of metastatic spread. METHODS: Resection of the mesopancreas (RMP) was performed in fresh corpses. Pancreas and mesopancreas were separated from each other and the mesopancreas was immunohistochemically investigated. RESULTS: The mesopancreas strains itself dorsally of the mesenteric vessels as a whitish-firm, fatty tissue-like layer. Macroscopically, in the dissected en-bloc specimens of pancreas and mesopancreas nerve plexuses were found running from the dorsal site of the pancreatic head to the mesopancreas to establish a perineural plane. Immunohistochemical examinations revealed the lymphatic vessels localized in direct vicinity of the neuronal plexuses between pancreas and mesopancreas. CONCLUSION: The mesopancreas as a perineural lymphatic layer located dorsally to the pancreas and reaching beyond the mesenteric vessels has not been classified in the anatomical or surgical literature before. The aim to ensure the greatest possible distance from the retropancreatic lymphatic tissue which drains the carcinomatous focus can be achieved in patients with pancreatic cancer only by complete resection of the mesopancreas (RMP).

The murine bronchopulmonary microcirculation in hapten-induced inflammation.

OBJECTIVE: The clinical observation of central bronchial artery hypertrophy in chronic lung inflammation suggests the possibility that the bronchial circulation may also participate in adaptive responses in peripheral lung inflammation. METHODS: To investigate the potential role of the bronchial microcirculation in peripheral lung inflammation, we developed a murine model of lung inflammation using the intratracheal instillation of the peptide-hapten trinitrophenol in presensitized mice. RESULTS: Clinical parameters indicated a peak inflammatory response at 96 hours. Similarly, gross and microscopic evidence of inflammation was observed 96 hours after antigen instillation. Using a forced oscillation technique to probe peripheral lung mechanics at 96 hours, we detected no change in central airway resistance (P > .05), but a significant increase in peripheral tissue resistance (P < .05). The structure of the bronchial circulation was investigated by microsphere occlusion of the pulmonary circulation and corrosion casting of the bronchial circulation. SEM of the bronchial artery casts demonstrated (1) the presence of the peripheral bronchial circulation in mice, (2) interconnections of the two systems in the distal bronchial arteries and at the level of alveolar capillaries, and (3) functional evidence of increased bronchial perfusion of alveolar capillaries during mononuclear inflammation. CONCLUSION: These results suggest an important adaptive role of the bronchial circulation in pulmonary inflammation.

Structural adaptations in the murine colon microcirculation associated with hapten-induced inflammation.

BACKGROUND: Blood flowing across the vascular endothelium creates wall shear stress, dependent on velocity of flow and vessel geometry, that tends to disrupt lymphocyte-endothelial cell adhesion. OBJECTIVE: The microcirculation in a murine model of acute colitis was investigated to identify structural adaptations during acute colitis that may facilitate transmigration. METHODS: In 2,4,6-trinitrobenzenesulphonic acid-induced acute colitis, the infiltrating cells and colonic microcirculation was investigated by cellular topographic mapping, corrosion casting and three-dimensional scanning electron microscopy (SEM). Colonic blood velocimetry was performed using intravital microscopy. RESULTS: Clinical and histological parameters suggested a peak inflammatory response at 96 h (p<0.001). The infiltrating cells were spatially related to the mucosal capillary plexus by three-dimensional topographic mapping (p<0.001). In normal mice, corrosion casting and three-dimensional SEM showed a polygonal mucosal plexus supplied by ascending arteries and descending veins. After 2,4,6-trinitrobenzenesulphonic acid stimulation, three-dimensional SEM showed preserved branch angles (p = 0.52) and nominal vessel lengths (p = 0.93), but a significantly dilated mucosal capillary plexus (p<0.001). Intravital microscopy of the mucosal plexus showed a greater than twofold decrease in the velocity of flow (p<0.001). CONCLUSIONS: The demonstrable slowing of the velocity of flow despite an increase in volumetric flow suggests that these microvascular adaptations create conditions suitable for leucocyte adhesion and transmigration.

Inflammation-responsive focal constrictors in the mouse ear microcirculation.

In many capillary exchange beds, blood flow is locally regulated by precapillary sphincter-like activity. In this study, we used intravascular tracers and scanning electron microscopy to investigate precapillary blood flow regulation in the mouse ear. Gelatin ink injections of the normal mouse ear demonstrated 6.8 +/- 2.3 axial vessels with a cutoff of detectable tracer in the early branches: 19 +/- 11 focal constrictions were observed along the 1st to 5th order branches of the axial vessels. A perfusion tracer consisting of biotinylated anti-endothelial lectins (Ricinus Communis Agglutin, Lycopersicon Esculentum and Griffonia Simplicifolia) was circulated for 30 min under physiological conditions. Subsequent enzyme histochemistry demonstrated no significant change in distal perfusion or in the number of focal constrictions (P > 0.05). Furthermore, the focal constrictions were unresponsive to vasodilators such as organic nitrates and prostaglandin E1. By contrast, the presence of oxazolone-induced inflammation resulted in significant and sustained vasodilatation for more than 96 h (P > 0.001). Scanning electron microscopy demonstrated discrete constricting bands morphologically distinct from known precapillary sphincters. These results suggest that these previously unappreciated inflammation-responsive precapillary constrictors regulate capillary recruitment in the mouse ear microcirculation.