CaMKII and MEK1/2 inhibition time-dependently modify inflammatory signaling in rat cerebral arteries during organ culture.

BACKGROUND: Cerebral ischemia induces transcriptional upregulation of inflammatory genes in the brain parenchyma and in cerebral arteries, thereby contributing to the infarct development. The present study was designed to evaluate the involvement of calcium-calmodulin-dependent protein kinase (CaMKII) II and extracellular signal-regulated kinase1/2 (ERK1/2) on inflammatory mediators in rat cerebral arteries using organ culture as a method for inducing ischemic-like vascular wall changes. METHODS: Rat basilar arteries were cultured in serum-free medium for 0, 3, 6 or 24 hours in the presence or absence of the CaMKII inhibitor KN93 or the MEK1/2 inhibitor U0126. Protein expression of activated CaMKII, ERK1/2, and inflammatory-associated protein kinases and mediators were examined with western blot and immunohistochemistry. Caspase-3 mRNA levels in basilar arteries were studied with real-time PCR. RESULTS: Western blot evaluation showed that organ culture induced a significant increase in phosphorylated ERK1/2 at 3, 6 and 24 hours, while CaMKII was found to be already activated in fresh non-incubated arteries and to decrease with incubation time. The addition of U0126 or KN93 decreased levels of phosphorylated c-Jun N-terminal kinase and p-p38, as evaluated by immunohistochemistry. KN93 affected the increase in caspase-3 mRNA expression only when given at the start of incubation, while U0126 had an inhibitory effect when given up to six hours later. Tumor necrosis factor receptor 1 was elevated after organ culture. This inflammatory marker was reduced by both of the two different protein kinase inhibitors. CONCLUSIONS: The novel findings of the present study are that the cross-talk between the two protein kinases and the inhibition of CaMKII or MEK1/2 in a time-dependent manner attenuates inflammatory-associated protein kinases and mediators, suggesting that they play a role in cerebrovascular inflammation.

Resolving uncertainty on the fly: modeling adaptive driving behavior as active inference.

Understanding adaptive human driving behavior, in particular how drivers manage uncertainty, is of key importance for developing simulated human driver models that can be used in the evaluation and development of autonomous vehicles. However, existing traffic psychology models of adaptive driving behavior either lack computational rigor or only address specific scenarios and/or behavioral phenomena. While models developed in the fields of machine learning and robotics can effectively learn adaptive driving behavior from data, due to their black box nature, they offer little or no explanation of the mechanisms underlying the adaptive behavior. Thus, generalizable, interpretable, computational models of adaptive human driving behavior are still rare. This paper proposes such a model based on active inference, a behavioral modeling framework originating in computational neuroscience. The model offers a principled solution to how humans trade progress against caution through policy selection based on the single mandate to minimize expected free energy. This casts goal-seeking and information-seeking (uncertainty-resolving) behavior under a single objective function, allowing the model to seamlessly resolve uncertainty as a means to obtain its goals. We apply the model in two apparently disparate driving scenarios that require managing uncertainty, (1) driving past an occluding object and (2) visual time-sharing between driving and a secondary task, and show how human-like adaptive driving behavior emerges from the single principle of expected free energy minimization.

The role of uncertainty and reward on eye movements in a virtual driving task.

Eye movements during natural tasks are well coordinated with ongoing task demands and many variables could influence gaze strategies. Sprague and Ballard (2003) proposed a gaze-scheduling model that uses a utility-weighted uncertainty metric to prioritize fixations on task-relevant objects and predicted that human gaze should be influenced by both reward structure and task-relevant uncertainties. To test this conjecture, we tracked the eye movements of participants in a simulated driving task where uncertainty and implicit reward (via task priority) were varied. Participants were instructed to simultaneously perform a Follow Task where they followed a lead car at a specific distance and a Speed Task where they drove at an exact speed. We varied implicit reward by instructing the participants to emphasize one task over the other and varied uncertainty in the Speed Task with the presence or absence of uniform noise added to the car's velocity. Subjects' gaze data were classified for the image content near fixation and segmented into looks. Gaze measures, including look proportion, duration and interlook interval, showed that drivers more closely monitor the speedometer if it had a high level of uncertainty, but only if it was also associated with high task priority or implicit reward. The interaction observed appears to be an example of a simple mechanism whereby the reduction of visual uncertainty is gated by behavioral relevance. This lends qualitative support for the primary variables controlling gaze allocation proposed in the Sprague and Ballard model.

Humans Use Similar Posture Sequences in a Whole-Body Tracing Task.

Humans have elegant bodies that allow gymnastics, piano playing, and tool use, but understanding how they do this in detail is difficult because their musculoskeletal systems are extremely complicated. Previous studies have shown that common movements such as reaching for a coffee cup, cycling a bicycle, or playing the piano have common patterns across subjects. This paper shows that an arbitrary set of whole-body movements used to trace large closed curves have common patterns both in the trajectory of the body's limbs and in variations within those trajectories. The commonality of the result should spur the search for explanations for its generality. One such principle could be that humans choose trajectories that are economical in energetic cost. Another synergistic possibility is that common movements can be saved in segments that can be combined to facilitate the process of deployment.

Excessive activation of poly(ADP-ribose) polymerase contributes to inherited photoreceptor degeneration in the retinal degeneration 1 mouse.

Retinitis pigmentosa (RP) is an inherited blinding disease for which there is no treatment available. It is characterized by a progressive and neurodegenerative loss of photoreceptors but the underlying mechanisms are poorly understood. Excessive activation of the enzyme poly(ADP-ribose) polymerase (PARP) has recently been shown to be involved in several neuropathologies. To investigate the possible role of PARP in retinal photoreceptor degeneration, we used the retinal degeneration 1 (rd1) mouse RP model to study PARP expression, PARP activity, and to test the effects of PARP inhibition on photoreceptor viability. PARP expression was found to be equal between rd1 and wild-type counterpart retinas. In contrast to this, a dramatic increase in both PARP activity per se and PARP product formation was detected by in situ assays in rd1 photoreceptors actively undergoing cell death. Furthermore, PARP activity colabeled with oxidatively damaged DNA and nuclear translocation of AIF (apoptosis-inducing factor), suggesting activation of PARP as a bridge between these events in the degenerating photoreceptors. The PARP-specific inhibitor PJ34 [N-(6-oxo-5,6-dihydrophenanthridin-2-yl)-N,N-dimethylacetamide x HCl[ reduced the number of cells exhibiting death markers in a short-term retinal culture paradigm, a protective effect that was translated into an increased number of surviving photoreceptors when the inhibitor was used in a long-term culture setting. Our results thus demonstrate an involvement of PARP activity in rd1 photoreceptor cell death, which could have a bearing on the understanding of neurodegenerations as such. The findings also suggest that the therapeutical possibilities of PARP inhibition should include retinal diseases like RP.

rd1 Mouse retina shows an imbalance in the activity of cysteine protease cathepsins and their endogenous inhibitor cystatin C.

PURPOSE: To compare in vivo levels, spatial localization, and in vitro secretion of cysteine protease cathepsins and cystatin C (cysC) in the retinal degeneration 1 (rd1) mouse model of retinitis pigmentosa and control (wt) mouse retinas. METHODS: The spatial localization, protein contents, cysC levels and cathepsin-B, -S, and -L activities in wt and rd1 retinas at postnatal (PN) days 2, 7, 14, 21, and 28 were analyzed by immunostaining, spectrophotometry, ELISA, and fluorescence spectrophotometry. The in vitro secretion of cysC and cysteine proteases by PN7 retinal explants into the conditioned medium (RCM) was quantified. RESULTS: The pigment epithelium, photoreceptors, and inner retinal and ganglion cell layers of both wt and rd1 retinas showed cysC and cathepsin-B labeling. CysC immunostaining was extensive in the optic nerve head fibers. The rd1 explants secreted higher amounts of cysteine protease into the RCM. The protein content in wt and rd1 retinal extracts increased up to PN14, then decreased in rd1 but not in wt. In rd1 extracts at PN14 to -28, cathepsin activity was higher and increased with age, but the cysC level was higher and constant. The ratios of cathepsin activity to cysC (cathepsin-L at PN2 and total, -B, and -L at PN14 to -28) were higher in rd1 extracts. CONCLUSIONS: Similar localization of both cathepsin-B and cysC in wt and rd1 retinas along with lower proteins and higher cathepsin activity in rd1 retinal extracts and RCM are consistent with their localization in extracellular matrix and a role in physiopathologic remodeling in wt and rd1 retinas.

CNTF+BDNF treatment and neuroprotective pathways in the rd1 mouse retina.

The rd1 mouse is a relevant model for studying the mechanisms of photoreceptor degeneration in retinitis pigmentosa. Treatment with ciliary neurotrophic factor (CNTF) in combination with brain derived neurotrophic factor (BDNF) is known to rescue photoreceptors in cultured rd1 retinal explants. To shed light on the underlying mechanisms, we studied the effects of 9 days (starting at postnatal day 2) in vitro CNTF+BDNF treatment on the endogenous production of CNTF, BDNF, fibroblast growth factor 2 (FGF2), or the activation of extracellular signal-regulated kinase (ERK), Akt and cAMP-response-element-binding protein (CREB) in retinal explants. In rd1 explants, CNTF+BDNF decreased the number of TUNEL-positive photoreceptors. The treatment also increased endogenous rd1 levels of CNTF and BDNF, but lowered the level of FGF2 expression in rd1 explants. When wild-type explants were treated, endogenous CNTF was similarly increased, while BDNF and FGF2 levels remained unaffected. In addition, treatment of rd1 retinas strongly increased the phosphorylation of ERK, Akt and CREB. In treated wild-type explants, the same parameters were either unchanged (ERK) or decreased (Akt and CREB). The results suggest a role for Akt, ERK and CREB in conveying the neuroprotective effect of CNTF+BDNF treatment in rd1 retinal explants.

Enhanced Endothelin-1 Mediated Vasoconstriction of the Ophthalmic Artery May Exacerbate Retinal Damage after Transient Global Cerebral Ischemia in Rat.

Cerebral vasculature is often the target of stroke studies. However, the vasculature supplying the eye might also be affected by ischemia. The aim of the present study was to investigate if the transient global cerebral ischemia (GCI) enhances vascular effect of endothelin-1 (ET-1) and 5-hydroxytryptamine/serotonin (5-HT) on the ophthalmic artery in rats, leading to delayed retinal damage. This was preformed using myography on the ophthalmic artery, coupled with immunohistochemistry and electroretinogram (ERG) to assess the ischemic consequences on the retina. Results showed a significant increase of ET-1 mediated vasoconstriction at 48 hours post ischemia. The retina did not exhibit any morphological changes throughout the study. However, we found an increase of GFAP and vimentin expression at 72 hours and 7 days after ischemia, indicating Müller cell mediated gliosis. ERG revealed significantly decreased function at 72 hours, but recovered almost completely after 7 days. In conclusion, we propose that the increased contractile response via ET-1 receptors in the ophthalmic artery after 48 hours may elicit negative retinal consequences due to a second ischemic period. This may exacerbate retinal damage after ischemia as illustrated by the decreased retinal function and Müller cell activation. The ophthalmic artery and ET-1 mediated vasoconstriction may be a valid and novel therapeutic target after longer periods of ischemic insults.

Predicting human visuomotor behaviour in a driving task.

The sequential deployment of gaze to regions of interest is an integral part of human visual function. Owing to its central importance, decades of research have focused on predicting gaze locations, but there has been relatively little formal attempt to predict the temporal aspects of gaze deployment in natural multi-tasking situations. We approach this problem by decomposing complex visual behaviour into individual task modules that require independent sources of visual information for control, in order to model human gaze deployment on different task-relevant objects. We introduce a softmax barrier model for gaze selection that uses two key elements: a priority parameter that represents task importance per module, and noise estimates that allow modules to represent uncertainty about the state of task-relevant visual information. Comparisons with human gaze data gathered in a virtual driving environment show that the model closely approximates human performance.

Glutathione preservation during storage of rat lenses in optisol-GS and castor oil.

BACKGROUND: Glutathione concentration in the lens decreases in aging and cataractous lenses, providing a marker for tissue condition. Experimental procedures requiring unfrozen lenses from donor banks rely on transportation in storage medium, affecting lens homeostasis and alterations in glutathione levels. The aim of the study was to examine the effects of Optisol-GS and castor oil on lens condition, determined from their ability to maintain glutathione concentrations. METHODOLOGY/PRINCIPAL FINDINGS: Rat lenses were stored in the two types of storage media at varying time intervals up to 3 days. Glutathione concentration was afterwards determined in an enzymatic detection assay, specific for both reduced and oxidized forms. Lenses removed immediately after death exhibited a glutathione concentration of 4.70±0.29 mM. In vitro stored lenses in Optisol-GS lost glutathione quickly, ending with a concentration of 0.60±0.34 mM after 3 days while castor oil stored lenses exhibited a slower decline and ended at 3 times the concentration. A group of lenses were additionally stored under post mortem conditions within the host for 6 hours before its removal. Total glutathione after 6 hours was similar to that of lenses removed immediately after death, but with altered GSH and GSSG concentrations. Subsequent storage of these lenses in media showed changes similar to those in the first series of experiments, albeit to a lesser degree. CONCLUSIONS/SIGNIFICANCE: It was determined that storage in Optisol-GS resulted in a higher loss of glutathione than lenses stored in castor oil. Storage for more than 12 hours reduced glutathione to half its original concentration, and was considered unusable after 24 hours.

Retinal adaptation to changing glycemic levels in a rat model of type 2 diabetes.

PURPOSE: Glucose concentrations are elevated in retinal cells in undiagnosed and in undertreated diabetes. Studies of diabetic patients suggest that retinal function adapts, to some extent, to this increased supply of glucose. The aim of the present study was to examine such adaptation in a model of type 2 diabetes and assess how the retina responds to the subsequent institution of glycemic control. METHODS: Electroretinography (ERG) was conducted on untreated Zucker diabetic fatty (ZDF) rats and congenic controls from 8-22 weeks of age and on ZDFs treated with daily insulin from 16-22 weeks of age. Retinal sections from various ages were prepared and compared histologically and by immunocytochemistry. PRINCIPAL FINDINGS/CONCLUSIONS: Acute hyperglycemia did not have an effect on control rats while chronic hyperglycemia in the ZDF was associated with scotopic ERG amplitudes which were up to 20% higher than those of age-matched controls. This change followed the onset of hyperglycemia with a delay of over one month, supporting that habituation to hyperglycemia is a slow process. When glycemia was lowered, an immediate decrease in ZDF photoreceptoral activity was induced as seen by a reduction in a-wave amplitudes and maximum slopes of about 30%. A direct effect of insulin on the ERG was unlikely since the expression of phosphorylated Akt kinase was not affected by treatment. The electrophysiological differences between untreated ZDFs and controls preceded an activation of Müller cells in the ZDFs (up-regulation of glial fibrillary acidic protein), which was attenuated by insulin treatment. There were otherwise no signs of cell death or morphological alterations in any of the experimental groups. These data show that under chronic hyperglycemia, the ZDF retina became abnormally sensitive to variations in substrate supply. In diabetes, a similar inability to cope with intensive glucose lowering could render the retina susceptible to damage.

The relationship between body and ambient temperature and corneal temperature.

PURPOSE: Exposure to elevated ambient temperatures has been mentioned as a risk factor for common eye diseases, primarily presbyopia and cataract. The aim of the present study was to examine the relationship among ambient, cornea, and body core temperature. METHODS: The relation between corneal temperature and ambient temperature was examined in 11 human volunteers. Furthermore, corneal temperature was measured using a thermal camera during elevation of body core temperature in three human volunteers and four rats. RESULTS: A linear relationship between corneal temperature and body temperature was found in the rat. For humans there was an initial linear increase in corneal temperature with increasing body temperature, but corneal temperature seemed to plateau at 36.5°C to 37.0°C despite a continued increase of body core temperature. A linear relationship between ambient and corneal temperature was found in humans but with a less steep slope than that between corneal and body core temperature. CONCLUSIONS: Corneal temperature is estimated to reach the maximum of 36.5°C to 37.0°C at ambient temperatures between 32.0°C and 34.5°C. If there is a causal relationship between elevated eye temperature, cataract, and presbyopia, the incidence of these eye diseases is predicted to increase with global warming. Importantly, the strong association between corneal temperature and body core temperature indicates that frequent infections could also be considered a risk factor for age-related lens disorders.

Calpain activity in retinal degeneration.

Retinal degenerations such as retinitis pigmentosa (RP) or glaucoma are a major cause of blindness in humans. Understanding the mechanisms underlying the various types of retinal degeneration is a pre-requisite for the development of rational therapies for these diseases. Activation of the calcium dependent protease, calpain, has been suggested to play an important role in cell death in various neuronal tissues including the retina. Improved detection and analysis of calpain activity during degenerative processes is likely to expand the list of pathological conditions with calpain involvement. We give a short overview of the methods available for the detection of calpain activity, and briefly discuss properties of calpain inhibitors. We then discuss the role of calpains in different cell death mechanisms and review existing work on retinal degeneration and the possible involvement of calpains therein. The implication of calpains in retinal cell death raises the possibility to use calpain inhibitors to prevent or delay retinal degeneration.

Differential Akt activation in the photoreceptors of normal and rd1 mice.

Retinitis pigmentosa is a blinding disease in which unknown mechanisms cause the degeneration of retinal photoreceptors. The retinal degeneration (rd1) mouse is a relevant model for this condition, since it carries a mutation also found in some forms of retinitis pigmentosa. To understand the degenerative process in the rd1 mouse, we must identify the survival and apoptosis-related signaling pathways in its photoreceptors and determine whether signaling differs from that in normal mice. The phosphatidylinositol 3-kinase/Akt kinase pathway promotes survival in several different cell types. The purpose of the present study has been to compare Akt activity in retinal cells of normal and rd1 mice. We have found that, in normal mice, Akt becomes activated in the retina in a developmentally regulated and cell-type-specific fashion, encompassing essentially all retinal cells. In most cell types, once Akt activation has begun, it remains in this state throughout life. An exception is seen in the rod photoreceptors, in which Akt is activated only transiently during their development. The rd1 retina behaves identically in all but one respect, namely that the activation of Akt in rod photoreceptors persists until these cells undergo apoptosis. Thus, Akt may participate in constitutive survival processes in retinal neurons, except in rod photoreceptors in which the role of this pathway may be restricted to the developmental period. However, Akt activation in the rods may be part of a defense mechanism initiated in response to insults, such as the retinal degeneration seen in the rd1 mouse.