David M. Wu

Second harmonic generating (SHG) nanoprobes for in vivo imaging

Fluorescence microscopy has profoundly changed cell and molecular biology studies by permitting tagged gene products to be followed as they function and interact. The ability of a fluorescent dye to absorb and emit light of different wavelengths allows it to generate startling contrast that, in the best cases, can permit single molecule detection and tracking. However, in many experimental settings, fluorescent probes fall short of their potential due to dye bleaching, dye signal saturation, and tissue autofluorescence. Here, we demonstrate that second harmonic generating (SHG) nanoprobes can be used for in vivo imaging, circumventing many of the limitations of classical fluorescence probes. Under intense illumination, such as at the focus of a laser-scanning microscope, these SHG nanocrystals convert two photons into one photon of half the wavelength; thus, when imaged by conventional two-photon microscopy, SHG nanoprobes appear to generate a signal with an inverse Stokes shift like a fluorescent dye, but with a narrower emission. Unlike commonly used fluorescent probes, SHG nanoprobes neither bleach nor blink, and the signal they generate does not saturate with increasing illumination intensity. The resulting contrast and detectability of SHG nanoprobes provide unique advantages for molecular imaging of living cells and tissues.

ATP: a vasoactive signal in the pericyte-containing microvasculature of the rat retina.

In this study we tested the hypothesis that extracellular ATP regulates the function of the pericyte‐containing retinal microvessels. Pericytes, which are more numerous in the retina than in any other tissue, are abluminally located cells that may adjust capillary perfusion by contracting and relaxing. At present, knowledge of the vasoactive molecules that regulate pericyte function is limited. Here, we focused on the actions of extracellular ATP because this nucleotide is a putative glial‐to‐vascular signal, as well as being a substance released by activated platelets and injured cells. In microvessels freshly isolated from the adult rat retina, we monitored ionic currents via perforated‐patch pipettes, measured intracellular calcium levels with the use of fura‐2, and visualized microvascular contractions with the aid of time‐lapse photography. We found that ATP induced depolarizing changes in the ionic currents, increased calcium levels and caused pericytes to contract. P2X7 receptors and UTP‐activated receptors mediated these effects. Consistent with ATP serving as a vasoconstrictor for the pericyte‐containing microvasculature of the retina, the microvascular lumen narrowed when an adjacent pericyte contracted. In addition, the sustained activation of P2X7 receptors inhibited cell‐to‐cell electrotonic transmission within the microvascular networks. Thus, ATP not only affects the contractility of individual pericytes, but also appears to regulate the spatial and temporal dynamics of the vasomotor response.

Effects of angiotensin II on the pericyte‐containing microvasculature of the rat retina

The aim of this study was to identify the mechanisms by which angiotensin II alters the physiology of the pericyte‐containing microvasculature of the retina. Despite evidence that this vasoactive signal regulates capillary perfusion by inducing abluminal pericytes to contract and thereby microvascular lumens to constrict, little is known about the events linking angiotensin exposure with pericyte contraction. Here, using microvessels freshly isolated from the adult rat retina, we monitored pericyte currents via perforated‐patch pipettes, measured pericyte calcium levels with fura‐2 and visualized pericyte contractions and lumen constrictions by time‐lapse photography. We found that angiotensin activates nonspecific cation (NSC) and calcium‐activated chloride channels; the opening of these channels induces a depolarization that is sufficient to activate the voltage‐dependent calcium channels (VDCCs) expressed in the retinal microvasculature. Associated with these changes in ion channel activity, intracellular calcium levels rise, pericytes contract and microvascular lumens narrow. Our experiments revealed that an influx of calcium through the NSC channels is an essential step linking the activation of AT1 angiotensin receptors with pericyte contraction. Although not required in order for angiotensin to induce pericytes to contract, calcium entry via VDCCs serves to enhance the contractile response of these cells. In addition to activating nonspecific cation, calcium‐activated chloride and voltage‐dependent calcium channels, angiotensin II also causes the functional uncoupling of pericytes from their microvascular neighbours. This inhibition of gap junction‐mediated intercellular communication suggests a previously unappreciated complexity in the spatiotemporal dynamics of the microvascular response to angiotensin II.

Physiology of rat retinal pericytes: modulation of ion channel activity by serum-derived molecules.

1 Pericytes, which are contractile cells located on the outer wall of microvessels, are thought to be particularly important in the retina where the ratio of these cells to vascular endothelial cells is the highest of any tissue. Retinal pericytes are of interest since they may regulate capillary blood flow and because their selective loss is an early event in diabetic retinopathy, which is a common sight‐threatening disorder associated with dysfunction of the blood‐retinal barrier. 2 Although a breakdown in the vascular endothelial barrier is a frequent pathophysiological event, knowledge of the effects of blood‐derived molecules on pericyte function is limited. Based on the premise that ion channels play a vital role in cellular function, we examined the effect of serum on the ionic currents of retinal pericytes. To do this, we used the perforated‐patch configuration of the patch‐clamp technique to monitor the whole‐cell currents of pericytes located on freshly isolated rat retinal microvessels. 3 Exposure to serum reversibly activated inward and outward currents in virtually all of the sampled retinal pericytes. Two types of sustained conductances were induced by serum. These were a calcium‐permeable non‐specific cation (NSC) current and a voltage‐dependent potassium current. In addition, exposure to serum increased the activity of chloride channels which caused transient depolarizing currents. 4 Associated with the activation of these conductances, the membrane potential showed a sustained decrease of 10 ± 2 mV from −56 mV to −46 mV and, also, transient depolarizations to near −30 mV. The serum‐induced depolarizations can activate the voltage‐gated calcium channels expressed by the retinal pericytes. 5 Calcium‐permeable NSC channels appear to play a critical role in the response of pericytes to serum‐derived molecules. Consistent with this, activation of the chloride and potassium channels was sensitive to SK&F 96365, which is a blocker of NSC channels. In addition, chloride and potassium channel activation was dependent on extracellular calcium. 6 The effects of serum on the activity of channels in retinal pericytes were qualitatively mimicked by insulin‐like growth factor‐1 (IGF‐1), which is a normal constituent of the blood. 7 There are significant differences in the effects of serum on retinal pericytes compared with vascular smooth muscle cells. Serum activated sustained conductances in retinal pericytes but not in the vascular smooth muscle cells. This suggests a fundamental difference in the mechanisms by which serum‐derived molecules affect these two types of cells. 8 We conclude that serum‐derived molecules, such as IGF‐1, can activate several types of ion channels in retinal pericytes. These changes in channel activity are likely to influence pericyte function at sites of a breakdown in the blood‐retinal barrier.

Dopamine activates ATP-sensitive K+ currents in rat retinal pericytes.

The relatively sparse vasculature of the retina minimizes obstruction to incoming light, but also poses a challenge to fulfilling the metabolic demands of retinal neurons. An efficient process for distributing energy supplies to areas of need is likely to involve neuron-derived vasoactive signals. However, knowledge of the mechanisms by which capillary perfusion is regulated by neuron-to-vascular signaling is limited. Potential targets of vasoactive molecules released from nerve cells are the pericytes, which are positioned on the endothelial walls of microvessels and are thought to play a role in controlling the microcirculation. In this study, we assessed the effect of dopamine on pericyte physiology. Because dopaminergic neurites are closely associated with microvessels that express dopamine receptors, this molecule is a putative neuron-to-capillary signal, as well as neurotransmitter. We used the perforated-patch configuration of the patch-clamp technique to monitor the whole-cell currents of pericytes located on microvessels freshly isolated from the adult rat retina. In 43% (58/134) of the sampled pericytes, we found that dopamine reversibly activated a hyperpolarizing current, which increased the membrane potential by 19 +/- 1 mV. This dopamine-induced current was inhibited by the ATP-sensitive potassium (KATP) channel blocker, glibenclamide. Consistent with a signaling pathway involving D1 dopamine receptors, adenylate cyclase and protein kinase A (PKA), the selective D1 antagonist, SCH23390, inhibited the hyperpolarizing effect of dopamine; the activator of adenylate cyclase, forskolin, mimicked the dopaminergic effect, and H89, which inhibits PKA, significantly reduced the hyperpolarization induced by dopamine. Taken together, our experiments indicate that a mechanism involving D1 dopamine receptors, adenylate cyclase, and PKA activates KATP currents in retinal pericytes. Our observations support the hypothesis that, in addition to being a neuromodulator, dopamine also serves as a signal linking neuronal activity with the function of the pericyte-containing microvasculature.

Electrotonic transmission within pericyte-containing retinal microvessels

Objective: Little is known about the electrotonic architecture of the pericyte‐containing retinal microvasculature. Here, the authors focus on the cell‐to‐cell transmission of hyperpolarization, which can induce abluminal pericytes to relax and lumens to dilate.

The electrotonic architecture of the retinal microvasculature: modulation by angiotensin II

Non‐technical summary  In the quest to understand how the circulatory system adjusts microvascular function to meet local metabolic demand, we focused on the retina whose circulatory system consists exclusively of microvessels. Since voltages induced by extracellular signals play a key role in generating vasomotor responses, we characterized the movement of voltage within the retinal microvasculature. To do this, we quantified voltage transmission between pairs of recording pipettes located at well‐defined sites in capillary/arteriole plexuses freshly isolated from the rat retina. We found that the retinal microvasculature is not simply a homogeneous syncytium, but has a complex electrotonic architecture with differing efficacies of voltage transmission. Furthermore, we discovered that the electrotonic architecture is not static, but is modulated by angiotensin. This newly appreciated action reveals that vasoactive signals can alter the functional organization of the microvasculature and, thereby, regulate the spatial extent of the circulatory systems response to voltage‐changing inputs.

Diabetes-induced inhibition of voltage-dependent calcium channels in the retinal microvasculature: role of spermine.

PURPOSE Although decentralized control of blood flow is particularly important in the retina, knowledge of the functional organization of the retinal microvasculature is limited. Here, the authors characterized the distribution and regulation of L-type voltage-dependent calcium channels (VDCCs) within the most decentralized operational complex of the retinal vasculature--the feeder vessel/capillary unit--which consists of a capillary network plus the vessel linking it with a myocyte-encircled arteriole. METHODS Perforated-patch recordings, calcium-imaging, and time-lapse photography were used to assess VDCC-dependent changes in ionic currents, intracellular calcium, abluminal cell contractility, and lumen diameter, in microvascular complexes freshly isolated from the rat retina. RESULTS Topographical heterogeneity was found in the distribution of functional VDCCs; VDCC activity was markedly greater in feeder vessels than in capillaries. Experiments showed that this topographical distribution occurs, in large part, because of the inhibition of capillary VDCCs by a mechanism dependent on the endogenous polyamine spermine. An operational consequence of functional VDCCs predominantly located in the feeder vessels is that voltage-driven vasomotor responses are generated chiefly in this portion of the feeder vessel/capillary unit. However, early in the course of diabetes, this ability to generate voltage-driven vasomotor responses becomes profoundly impaired because of the inhibition of feeder vessel VDCCs by a spermine-dependent mechanism. CONCLUSIONS The regulation of VDCCs by endogenous spermine not only plays a critical role in establishing the physiological organization of the feeder vessel/capillary unit, but also may contribute to dysfunction of this decentralized operational unit in the diabetic retina.

Long-term follow-up of a family with dominant X-linked retinitis pigmentosa

PurposeTo document the progression of disease in male and female members of a previously described family with X-linked dominant retinitis pigmentosa (RP) caused by a de novoinsertion after nucleotide 173 in exon ORF15 of RPGR.MethodsThe clinical records of 19 members of family UTAD054 were reviewed. Their evaluations consisted of confirmation of family history, standardised electroretinograms (ERGs), Goldmann visual fields, and periodic ophthalmological examinations over a 23-year period.ResultsMale members of family UTAD054 had non-recordable to barely recordable ERGs from early childhood. The males showed contracted central fields and developed more severe retinopathy than the females. The female members showed a disease onset delayed to teenage years, recordable but diminishing photopic and scotopic ERG amplitudes in a cone-rod pattern, progressive loss and often asymmetric visual fields, and diffuse atrophic retinopathy with fewer pigment deposits compared with males.ConclusionsThis insertion mutation in the RPGRexon ORF15 is associated with a RP phenotype that severely affects males early and females by 30 years of age, and is highly penetrant in female members. Families with dominant-acting RPGRmutations may be mistaken to have an autosomal mode of inheritance resulting in an incorrect prediction of recurrence risk and prognosis. Broader recognition of X-linked RP forms with dominant inheritance is necessary to facilitate appropriate counselling of these patients.

Ocriplasmin for Treatment of Stage 2 Macular Holes: Early Clinical Results

BACKGROUND AND OBJECTIVE To review clinical and structural outcomes of ocriplasmin for treatment of stage 2 macular holes. PATIENTS AND METHODS A retrospective review of the first patients with stage 2 macular holes to be treated with ocriplasmin at Massachusetts Eye and Ear Infirmary. All patients were imaged with spectral-domain optical coherence tomography (SD-OCT). RESULTS Eight patients with stage 2 macular holes received a single injection of 125 μg of ocriplasmin. One patient (12.5%) demonstrated macular hole closure. The posterior hyaloid separated from the macula in six eyes (75%). All seven holes that remained open showed enlargement in hole diameters (narrowest, apical, and basal) at 1 week and 1 month. All seven were successfully closed with surgery. Ellipsoid zone disruptions were observed by OCT in four eyes (50%) and persisted throughout follow-up (more than 6 months on average). CONCLUSION In early clinical results, the authors found a lower macular hole closure rate with ocriplasmin than previously reported. Enlargement was observed in all holes that failed to close with ocriplasmin. The authors found ellipsoid zone disruptions that persisted through 6 months of follow-up after ocriplasmin injection. Further work is needed to investigate the cause for these ellipsoid zone changes.