Clemens Strohmaier

Brain and Retinal Pericytes: Origin, Function and Role

Pericytes are specialized mural cells located at the abluminal surface of capillary blood vessels, embedded within the basement membrane. In the vascular network these multifunctional cells fulfil diverse functions, which are indispensable for proper homoeostasis. They serve as microvascular stabilizers, are potential regulators of microvascular blood flow and have a central role in angiogenesis, as they for example regulate endothelial cell proliferation. Furthermore, pericytes, as part of the neurovascular unit, are a major component of the blood-retina/brain barrier. CNS pericytes are a heterogenic cell population derived from mesodermal and neuro-ectodermal germ layers acting as modulators of stromal and niche environmental properties. In addition, they display multipotent differentiation potential making them an intriguing target for regenerative therapies. Pericyte-deficiencies can be cause or consequence of many kinds of diseases. In diabetes, for instance, pericyte-loss is a severe pathological process in diabetic retinopathy (DR) with detrimental consequences for eye sight in millions of patients. In this review, we provide an overview of our current understanding of CNS pericyte origin and function, with a special focus on the retina in the healthy and diseased. Finally, we highlight the role of pericytes in de- and regenerative processes.

Long-term outcomes after monocular corneal inlay implantation for the surgical compensation of presbyopia

Purpose To evaluate long‐term outcomes of small‐aperture corneal inlay implantation for the surgical compensation of presbyopia. Setting Paracelsus Medical University, Salzburg, Austria. Design Prospective interventional cohort study. Methods Monocular implantation of a Kamra small‐aperture inlay (model ACI7000) (1.6 mm central aperture) was performed in emmetropic presbyopic eyes. The preoperative and postoperative parameters included monocular and binocular uncorrected (UDVA) and corrected (CDVA) distance visual acuities, uncorrected intermediate visual acuity (UIVA), and uncorrected (UNVA) and corrected (CNVA) near visual acuities; refraction; patient satisfaction; and complications. Results From September 4, 2006, to May 21, 2007, a small‐aperture inlay (1.6 mm central aperture) was implanted in 32 emmetropic presbyopic eyes. The mean binocular uncorrected visual acuities improved as follows: UNVA from Jaeger (J) 6 ± 1.2 lines (˜20/50) to J2 ± 1.8 lines (˜20/25) (P < .001) and UIVA from 0.2 logMAR ± 1.3 lines (˜20/32) to 0.1 logMAR ± 1.3 lines (˜20/25) (P = .04). The UDVA decreased from −0.2 logMAR ± 0.2 lines (˜20/12.5) to −0.1 logMAR ± 0.6 lines (˜20/16) (P < .001). At 60 months, 74.2% of patients had a UNVA of J3 (˜20/32) or better, 87.1% had a UIVA of 0.2 logMAR (˜20/32) or better, and 93.5% had a UDVA of 0.0 logMAR (˜20/20) or better. One inlay was removed after 36 months because of patient dissatisfaction with vision after a hyperopic shift in the surgical eye, with no loss of CDVA or CNVA 2 years after removal. Conclusion Long‐term results of monocular corneal inlay implantation indicate increased UNVA and UIVA and slightly compromised UDVA in emmetropic presbyopic eyes. Financial Disclosure Dr. Grabner was reimbursed for travel expenses from Acufocus. Dr. Riha is a consultant to Acufocus. No other author has a financial or proprietary interest in any material or method mentioned.

A New Nanosecond UV Laser at 355 nm: Early Results of Corneal Flap Cutting in a Rabbit Model

PURPOSE A new 355 nm UV laser was used for corneal flap cutting in an animal model and tested for clinical and morphologic alterations. METHODS Corneal flaps were created (Chinchilla Bastards; n = 25) with an UV nanosecond laser at 355 nm (150 kHz, pulse duration 850 ps, spot-size 1 μm, spot spacing 6 × 6 μm, side cut Δz 1 μm; cutting depth 130 μm) and pulse energies of 2.2 or 2.5 μJ, respectively. Following slit-lamp examination, animals were killed at 6, 12, and 24 hours after treatment. Corneas were prepared for histology (hematoxylin and eosin [HE], TUNEL-assay) and evaluated statistically, followed by ultrastructural investigations. RESULTS Laser treatment was tolerated well, flap lift was easier at 2.5 μJ compared with 2.2 μJ. Standard HE at 24 hours revealed intact epithelium in the horizontal cut, with similar increase in corneal thickness at both energies. Irrespective of energy levels, TUNEL assay revealed comparable numbers of apoptotic cells in the horizontal and vertical cut at 6, 12, and 24 hours, becoming detectable in the horizontal cut as an acellular stromal band at 24 hours. Ultrastructural analysis revealed regular morphology in the epi- and endothelium, while in the stroma, disorganized collagen lamellae were detectable representing the horizontal cut, again irrespective of energy levels applied. CONCLUSIONS This new UV laser revealed no epi- nor endothelial damage at energies feasible for corneal flap cutting. Observed corneal swelling was lower compared with existing UV laser studies, albeit total energy applied here was much higher. Observed loss of stromal keratinocytes is comparable with available laser systems. Therefore, this new laser is suitable for refractive surgery, awaiting its test in a chronic environment.

Novel non-contact retina camera for the rat and its application to dynamic retinal vessel analysis

We present a novel non-invasive and non-contact system for reflex-free retinal imaging and dynamic retinal vessel analysis in the rat. Theoretical analysis was performed prior to development of the new optical design, taking into account the optical properties of the rat eye and its specific illumination and imaging requirements. A novel optical model of the rat eye was developed for use with standard optical design software, facilitating both sequential and non-sequential modes. A retinal camera for the rat was constructed using standard optical and mechanical components. The addition of a customized illumination unit and existing standard software enabled dynamic vessel analysis. Seven-minute in-vivo vessel diameter recordings performed on 9 Brown-Norway rats showed stable readings. On average, the coefficient of variation was (1.1 ± 0.19) % for the arteries and (0.6 ± 0.08) % for the veins. The slope of the linear regression analysis was (0.56 ± 0.26) % for the arteries and (0.15 ± 0.27) % for the veins. In conclusion, the device can be used in basic studies of retinal vessel behavior.

Intraindividual aqueous flare comparison after implantation of hydrophobic intraocular lenses with or without a heparin-coated surface

Purpose To assess the efficacy of a heparin‐surface‐modified (HSM) hydrophobic acrylic intraocular lens (IOL) (EC‐1YH PAL) and the same IOL without heparin coating (EC‐1Y‐PAL) by the flare and cell intensity in the anterior chamber after uneventful cataract surgery. Setting Department of Ophthalmology, Paracelsus Medical University Salzburg, Austria. Design Comparative case series. Methods Routine phacoemulsification with randomized implantation of an HSM IOL in 1 eye (HSM IOL group) and an uncoated IOL (uncoated IOL group) in the fellow eye was performed. Postoperative inflammation was assessed objectively using a laser flare–cell meter (FM‐600) preoperatively as well as 1 day and 1 and 3 months postoperatively. Aqueous cells in the anterior chamber, distance visual acuities, and subjective manifest refraction were also evaluated at each visit. Results One hundred eyes (50 patients) were enrolled. In both groups, the mean flare values increased significantly from preoperatively to 1 day postoperatively (P<.001) and nearly reached preoperative values by 3 months postoperatively. One day postoperatively, the mean flare value was statistically significantly lower in the HSM IOL group (14.92 photons per millisecond [ph/ms] ± 7.47 [SD]) than in the uncoated IOL group (mean 16.73 ± 7.81 ph/ms) (P=.04); there was no statistically significant difference between groups 1 and 3 months postoperatively (both P > .58). The HSM IOL group had a greater and quicker decrease in aqueous cells, reaching statistical significance 1 month postoperatively (P=.01). Conclusion The HSM IOL showed a significant lower inflammatory reaction in the early postoperative stage with a faster disappearance of inflammatory signs. Financial Disclosure No author has a financial or proprietary interest in any material or method mentioned.

The effect of vasopressin on choroidal blood flow, intraocular pressure, and orbital venous pressure in rabbits.

PURPOSE To investigate the effects of arginine-vasopressin (AVP) on intraocular pressure (IOP), orbital venous pressure (OVP), and choroidal blood flow (ChorBF) regulation in anesthetized rabbits. METHODS Mean arterial pressure (MAP), IOP, and OVP were measured by direct cannulation of the central ear artery, the vitreous, and the orbital venous sinus, respectively. Laser Doppler flowmetry was used to record ChorBF. To change the perfusion pressure (PP), MAP was manipulated mechanically with occluders around the aorta and vena cava. In the first group of animals (n = 11) the dose-response relationship was measured. In the second group of animals (n = 8) pressure-flow relationships were determined at baseline and in response to intravenous application of a low (0.08 ng/kg/min) and a high (1.33 ng/kg/min) infusion rate of AVP. RESULTS AVP caused a dose-dependent increase of MAP and choroidal vascular resistance (ChorR), whereas IOP, OVP, ChorBF, and heart rate (HR) were decreased. In contrast to the high infusion rate, the low infusion rate of AVP had no effect on baseline ChorBF. However, the pressure-flow relationship was shifted downward significantly by both infusion rates at PP below baseline. CONCLUSIONS AVP reduces IOP and OVP significantly and is a potent vasoconstrictor in the choroidal vascular bed. In the choroid, the effect of AVP is not only dose-dependent, but also PP-dependent, which is indicated by the reduced perfusion relative to control with low-dosed AVP at low PP.

Rat choroidal pericytes as a target of the autonomic nervous system

Pericytes are contractile cells that surround blood vessels. When contracting, they change the diameter of the vessel and therefore influence blood flow homeostasis; however, mechanisms controlling pericyte action are less well understood. Since blood flow regulation per se is controlled by the autonomic nervous system, the latter might also be involved in pericyte action. Hence, rat choroidal pericytes were analyzed for such a connection by using appropriate markers. Rat choroidal wholemounts and sections were prepared for immunohistochemistry of the pericyte marker chondroitin-sulfate-proteoglycan (NG2) and the pan-neuronal marker PGP9.5 or of tyrosine hydroxylase (TH), vasoactive intestinal polypeptide (VIP) and choline acetyl transferase (ChAT). Additionally, PGP9.5 and TH were analyzed in the choroid of DCX-dsRed2 transgenic rats, displaying red-fluorescent perivascular cells and serving as a putative model for studying pericyte function in vivo. Confocal laser-scanning microscopy revealed NG2-immunoreactive cells and processes surrounding the blood vessels. These NG2-positive cells were not co-localized with PGP9.5 but received close appositions of PGP9.5-, TH-, VIP- and ChAT-immunoreactive boutons and fibers. In the DCX-dsRed2 transgenic rat, PGP9.5 and TH were also densely apposed on the dsRed-positive cells adjacent to blood vessels. These cells were likewise immunoreactive for NG2, suggesting their pericyte identity. In addition to the innervation of vascular smooth muscle cells, the close relationship of PGP9.5 and further sympathetic (TH) and parasympathetic (VIP, ChAT) nerve fibers on NG2-positive pericytes indicated an additional target of the autonomic nervous system for choroidal blood flow regulation. Similar findings in the DCX-dsRed transgenic rat indicate the potential use of this animal model for in vivo experiments revealing the role of pericytes in blood flow regulation.

Episcleral venous pressure and IOP responses to central electrical stimulation in the rat.

PURPOSE Histological evidence suggests a role for the central nervous system in controlling episcleral venous pressure (EVP). Based on prior studies that identified candidate regions in the brain stem, the present study assessed the effect of electrical stimulation at the location of the superior salivatory nucleus (SSN) on EVP in rats. METHODS Male Sprague-Dawley rats (n = 11) were anesthetized using pentobarbital sodium (50 mg/kg intraperitoneally initially, supplemented intravenously [IV] as needed) and paralyzed with gallamine triethiodide (1 mg/kg, IV). The animals were artificially ventilated and the femoral artery and vein were cannulated for blood pressure measurement and drug administration. Carotid blood flow was measured with an ultrasound flow probe and heart rate with a cardiotachometer. IOP was measured through a cannula in the vitreous compartment and EVP was measured through a micropipette in episcleral veins using the servonull technique. After a craniotomy was performed, a unipolar stainless steel electrode was inserted into the brainstem at the coordinates of the SSN using a stereotactic instrument. Stimulations were performed at 20Hz, 9 μA, 1 ms pulse duration, and 200 pulses. RESULTS Stimulation at the SSN coordinates increased IOP from 10.6 ± 0.4 to 11.8 ± 0.6 mm Hg (P < 0.01) and EVP from 7.8 ± 1.3 to 10.7 ± 1.1 mm Hg (P < 0.01). Mean arterial pressure, carotid blood flow, and heart rate remained unaltered. CONCLUSIONS The present study indicates that the SSN may participate in regulating EVP.

Vector Analysis, Rotational Stability, and Visual Outcome After Implantation of a New Aspheric Toric IOL

PURPOSE To evaluate vector analysis, rotational stability, and refractive and visual outcome of a new toric intraocular lens (IOL) for correction of preexisting corneal astigmatism during routine cataract surgery. METHODS In this prospective, interventional case series, 30 toric, aspheric Bi-Flex T toric IOLs (Medicontur Medical Engineering Ltd., Inc., Zsámbék, Hungary) were implanted in 20 consecutive patients with topographic corneal astigmatism between 1.50 and 4.00 diopters (D) and evaluated within the first year after implantation. Appropriate IOL-toric alignment was facilitated by combined imaging/eye tracking technology. Postoperative evaluation included refraction and uncorrected and corrected distance visual acuities (UDVA, CDVA). For each visit, photodocumentation in retroillumination was performed to evaluate toric alignment and potential toric IOL rotation. Vector analysis of refractive astigmatism was performed using the Alpins method. RESULTS At 12 months postoperatively, a reduction of the refractive astigmatism from 1.93±0.90 D (range: 0.50 to 4.00 D) to 0.28±0.61 D (range: 0.00 to 1.50 D) could be found, with patients achieving a mean UDVA of 0.06±0.16 logMAR (range: -0.18 to 0.40 logMAR; Snellen 20/20). Intraoperative to 12-month postoperative comparison of IOL axis alignment showed low levels of rotation (0.2°±2.41°; range: +4° to -5°). Vector analysis showed target induced astigmatism of 0.60 D @180°, surgically induced astigmatism of 0.80 D @177°, correction index of 1.02±0.25, and a difference vector of 0.30 D @82°. CONCLUSIONS Implantation of the new Bi-Flex T IOL was a safe, stable, and effective method to correct preexisting regular corneal astigmatism during cataract surgery.

Distribution of the regulatory peptide alarin in the eye of various species

Alarin is a recently discovered regulatory peptide with vasoconstrictive properties in murine skin. Control of vasoconstriction/-relaxation is essential for ocular blood flow and hence the eyes homeostasis, and regulatory peptides are involved in regulation of ocular blood flow. Here we describe the existence and distribution of alarin in the eye of human and potential experimental animals (rat, mouse). Eyes of rat, mouse, and human were prepared for immunohistochemistry against murine and human alarin, respectively. Additionally, double staining experiments for alarin and CD31 were performed in human choroidal flat-mount preparations. For documentation, confocal laser scanning microscopy was used while quantitative real-time-PCR was applied to confirm immunohistochemical data and to detect alarin mRNA expression in human retina and choroid. Alarin-like immunoreactivity (alarin-LI) was detected in corneal epi- and endothelium of human, mouse, and rat, as well as in the conjunctiva of mouse and rat. Alarin-LI was found in the iris of all the species investigated and, in humans, was concentrated around blood vessels. All three species showed distinctive alarin-LI in the non-pigmented epithelium of the ciliary body. In the retina of mouse and rat, maximum signals were detected in the outer nuclear and ganglion cell layer, whereas in humans a strong alarin-LI was found around retinal blood vessels and in intrinsic choroidal neurons (ICN). Quantitative RT-PCR in human confirmed alarin mRNA expression retina and choroid. The existence of alarin in cornea and conjunctiva might indicate a role in immune defense, while its presence in the non-pigmented ciliary epithelium favors an involvement in aqueous humor production. Alarin around blood vessels/in ICN might indicate an involvement in ocular blood flow regulation. Since alarin is found widely distributed in the eyes of species investigated, we were able to establish the basis for further functional experiments.