Luis Alarcón-Martínez

Understanding glaucomatous damage: Anatomical and functional data from ocular hypertensive rodent retinas

Glaucoma, the second most common cause of blindness, is characterized by a progressive loss of retinal ganglion cells and their axons, with a concomitant loss of the visual field. Although the exact pathogenesis of glaucoma is not completely understood, a critical risk factor is the elevation, above normal values, of the intraocular pressure. Consequently, deciphering the anatomical and functional changes occurring in the rodent retina as a result of ocular hypertension has potential value, as it may help elucidate the pathology of retinal ganglion cell degeneration induced by glaucoma in humans. This paper predominantly reviews the cumulative information from our laboratorys previous, recent and ongoing studies, and discusses the deleterious anatomical and functional effects of ocular hypertension on retinal ganglion cells (RGCs) in adult rodents. In adult rats and mice, perilimbar and episcleral vein photocauterization induces ocular hypertension, which in turn results in devastating damage of the RGC population. In wide triangular sectors, preferentially located in the dorsal retina, RGCs lose their retrograde axonal transport, first by a functional impairment and after by mechanical causes. This axonal damage affects up to 80% of the RGC population, and eventually causes their death, with somal and intra-retinal axonal degeneration that resembles that observed after optic nerve crush. Importantly, while ocular hypertension affects the RGC population, it spares non-RGC neurons located in the ganglion cell layer of the retina. In addition, functional and morphological studies show permanent alterations of the inner and outer retinal layers, indicating that further to a crush-like injury of axon bundles in the optic nerve head there may by additional insults to the retina, perhaps of ischemic nature.

Ocular hypertension impairs optic nerve axonal transport leading to progressive retinal ganglion cell degeneration.

Ocular hypertension (OHT) is the main risk factor of glaucoma, a neuropathy leading to blindness. Here we have investigated the effects of laser photocoagulation (LP)-induced OHT, on the survival and retrograde axonal transport (RAT) of adult rat retinal ganglion cells (RGC) from 1 to 12 wks. Active RAT was examined with fluorogold (FG) applied to both superior colliculi (SCi) 1 wk before processing and passive axonal diffusion with dextran tetramethylrhodamine (DTMR) applied to the optic nerve (ON) 2 d prior to sacrifice. Surviving RGCs were identified with FG applied 1 wk pre-LP or by Brn3a immunodetection. The ON and retinal nerve fiber layer were examined by RT97-neurofibrillar staining. RGCs were counted automatically and color-coded density maps were generated. OHT retinas showed absence of FG+ or DTMR+RGCs in focal, pie-shaped and diffuse regions of the retina which, by two weeks, amounted to, approximately, an 80% of RGC loss without further increase. At this time, there was a discrepancy between the total number of surviving FG-prelabelled RGCs and of DMTR+RGCs, suggesting that a large proportion of RGCs had their RAT impaired. This was further confirmed identifying surviving RGCs by their Brn3a expression. From 3 weeks onwards, there was a close correspondence of DTMR+RGCs and FG+RGCs in the same retinal regions, suggesting axonal constriction at the ON head. Neurofibrillar staining revealed, in ONs, focal degeneration of axonal bundles and, in the retinal areas lacking backlabeled RGCs, aberrant staining of RT97 characteristic of axotomy. LP-induced OHT results in a crush-like injury to ON axons leading to the anterograde and protracted retrograde degeneration of the intraocular axons and RGCs.

Retinal ganglion cell population in adult albino and pigmented mice: A computerized analysis of the entire population and its spatial distribution

UNLABELLED In adult Swiss albino and C57 pigmented mice, RGCs were identified with a retrogradely transported neuronal tracer applied to both optic nerves (ON) or superior colliculi (SCi). After histological processing, the retinas were prepared as whole-mounts, examined and photographed under a fluorescence microscope equipped with a motorized stage controlled by a commercial computer image analysis system: Image-Pro Plus((R)) (IPP). Retinas were imaged as a stack of 24-bit color images (140 frames per retina) using IPP with the Scope-Pro plug-in 5.0 and the images montaged to create a high-resolution composite of the retinal whole-mount when required. Single images were also processed by specific macros written in IPP that apply a sequence of filters and transformations in order to separate individual cells for automatic counting. Cell counts were later transferred to a spreadsheet for statistical analysis and used to generate a RGC density map for each retina. RESULTS The mean total numbers of RGCs labeled from the ON, in Swiss (49,493+/-3936; n=18) or C57 mice (42,658+/-1540; n=10) were slightly higher than the mean numbers of RGCs labeled from the SCi, in Swiss (48,733+/-3954; n=43) or C57 mice (41,192+/-2821; n=42), respectively. RGCs were distributed throughout the retina and density maps revealed a horizontal region in the superior retina near the optic disk with highest RGC densities. In conclusion, the population of mice RGCs may be counted automatically with a level of confidence comparable to manual counts. The distribution of RGCs adopts a form of regional specialization that resembles a horizontal visual streak.

Quantification of the Effect of Different Levels of IOP in the Astroglia of the Rat Retina Ipsilateral and Contralateral to Experimental Glaucoma

PURPOSE To analyze the effects of different levels of intraocular pressure (IOP) in the macroglia in ocular hypertension (OHT) and contralateral eyes at 3 weeks after laser photocoagulation and compare these with effects in age-matched control rats. METHODS Adult Sprague-Dawley rats were divided into an age-matched control (naive) group and an OHT group. Retinas were processed as whole mounts and immunostained with GFAP for analysis of the retinal macroglia. RESULTS The area of the retina occupied by astrocytes (AROA) was quantified. GFAP immunostaining showed common features in ipsilateral and contralateral eyes. First, although the astrocyte network maintained a star-shaped morphology, these cells had fewer secondary processes and thinner cell bodies and primary processes than did naive cells. Second, Müller cells appeared as punctate GFAP+ structures among astrocytes. Third, there was a significant reduction of the AROA in ipsilateral and contralateral eyes compared with naive eyes. Ipsilateral eyes had significantly less AROA than did contralateral eyes. The decrease was greater for OHT eyes with higher IOP levels. CONCLUSIONS OHT induces changes in the macroglia of contralateral eyes; thus, these fellow eyes should not be used as control. In eyes with OHT, there is a close relationship between IOP values and decreased AROA.

Changes in the inner and outer retinal layers after acute increase of the intraocular pressure in adult albino Swiss mice

In adult albino mice the effects of increased intraocular pressure on the outer retina and its circuitry was investigated at intervals ranging 3-14 weeks. Ocular hypertension (OHT) was induced by cauterizing the vessels draining the anterior part of the mice eye, as recently reported (Salinas-Navarro et al., 2009a). Electroretinographic (ERG) responses were recorded simultaneously from both eyes and compared each other prior to and at different survival intervals of 2, 8 or 12 weeks after lasering. Animals were processed at 3, 9 or 14 weeks after lasering, and radial sections were obtained in the cryostat and further processed for immunocytochemistry using antibodies against recoverin, gamma-transducin, Protein Kinase C-alpha (PKC-alpha), calbindin or synaptophysin. The synaptic ribbons were identified using an antibody against the protein bassoon, which labels photoreceptor ribbons and nuclei were identified using TO-PRO. Laser photocoagulation of the perilimbar and episcleral veins of the left eye resulted in an increase in mean intraocular pressure to approximately over twice its baseline by 24 h that was maintained for approximately five days reaching basal levels by 1 week. ERG recordings from the different groups of mice showed their a-, b-wave and scotopic threshold response (STR) amplitudes, when compared to their contralateral fellow eye, reduced to 62%, 52% and 23% at 12 weeks after lasering. Three weeks after lasering, immunostaining with recoverin and transducin antibodies could not document any changes in the outer nuclear layer (ONL) but both ON-rod bipolar and horizontal cells had lost their dendritic processes in the outer plexiform layer (OPL). Sprouting of horizontal and bipolar cell processes were observed into the ONL. Fourteen weeks after lasering, protein kinase-C antibodies showed morphologic changes of ON-rod bipolar cells and calbindin staining showed abnormal horizontal cells and a loss of their relationship with their presynaptic input. Moreover, at this time, quantitative studies indicate significant diminutions in the number of photoreceptor synaptic ribbons/100 microm, and in the thickness of the outer nuclear and plexiform layer, when compared to their fellow eyes. Increased intraocular pressure in Swiss mice results in permanent alterations of their full field ERG responses and in changes of the inner and outer retinal circuitries.

Automated Quantification and Topographical Distribution of the Whole Population of S- and L-Cones in Adult Albino and Pigmented Rats

PURPOSE To quantify the whole population of S- and L-cones in the albino (Sprague-Dawley, SD) and pigmented (Piebald Virol Glaxo, PVG) rats and to study their topographical distribution within the retina. METHODS Retinal radial sections and whole-mounted retinas were double immunodetected with antibodies against UV-sensitive and L-opsins to detect the S- and L-cones, respectively. Two automated routines were developed to quantify the whole population of S- and L-cones. Detailed isodensity maps of each cone type were generated. In both strains, the presence of dual cones was detected, these were semiautomatically quantified and their distribution determined. The matching distribution of retinal ganglion cells (RGCs) and L-cones was attained by double immunodetection of Brn3a and L-opsin, respectively. RESULTS The mean number +/- SEM of L- or S-cones in SD and PVG retinas was 231,736 +/- 14,517 and 239,939 +/- 6,494 or 41,028 +/- 5,074, and 27,316 +/- 2,235, respectively. There was an increasing gradient of S-cone density along the inferonasal quadrant, although the highest densities were found in the retinal rims. The distribution of L-cones seemed to be complementary to the S-cones. The highest densities were observed in the superior nasotemporal axis, paralleling the distribution of Brn3a-positive RGCs. CONCLUSIONS These data establish, for the first time, the total number and the topographical distribution of S- and L-cones in two rat strains and demonstrate the correlation of L-cones and RGC spatial distribution.

Time-course of the retinal nerve fibre layer degeneration after complete intra-orbital optic nerve transection or crush: A comparative study

We examined qualitatively and quantitatively in adult rat retinas the temporal degeneration of the nerve fibre layer after intra-orbital optic nerve transection (IONT) or crush (IONC). Retinal ganglion cell (RGC) axons were identified by their heavy neurofilament subunit phosphorylated isoform (pNFH) expression. Optic nerve injury induces a progressive axonal degeneration which after IONT proceeds mainly with abnormal pNFH-accumulations in RCG axons and after IONC in RGCs somas and dendrites. Importantly, this aberrant pNFH-expression pattern starts earlier and is more dramatic after IONT than after IONC, highlighting the importance that the type of injury has on the time-course of RGC degeneration.

ERG changes in albino and pigmented mice after optic nerve transection

Optic nerve transection (ONT) triggers retinal ganglion cell (RGC) death. By using this paradigm, we have analyzed for the first time in adult albino and pigmented mice, the effects of ONT in the scotopic threshold response (STR) components (negative and positive) of the full-field electroretinogram. Two weeks after ONT, when in pigmented mice approximately 18% of the RGC population survive, the STR-implicit time decreased and the p and nSTR waves diminished approximately to 40% or 55%, in albino or pigmented, respectively, with respect to the values recorded from the non-operated contralateral eyes. These changes were maintained up to 12 weeks post-ONT, demonstrating that the ERG-STR is a useful parameter to monitor RGC functionality in adult mice.

A Novel In Vivo Model of Focal Light Emitting Diode-Induced Cone-Photoreceptor Phototoxicity: Neuroprotection Afforded by Brimonidine, BDNF, PEDF or bFGF

We have investigated the effects of light-emitting diode (LED)-induced phototoxicity (LIP) on cone-photoreceptors and their protection with brimonidine (BMD), brain-derived neurotrophic factor (BDNF), pigment epithelium-derived factor (PEDF), ciliary neurotrophic factor (CNTF) or basic fibroblast growth factor (bFGF). In anesthetized, dark adapted, adult albino rats a blue (400 nm) LED was placed perpendicular to the cornea (10 sec, 200 lux) and the effects were investigated using Spectral Domain Optical Coherence Tomography (SD-OCT) and/or analysing the retina in oriented cross-sections or wholemounts immune-labelled for L- and S-opsin and counterstained with the nuclear stain DAPI. The effects of topical BMD (1%) or, intravitreally injected BDNF (5 µg), PEDF (2 µg), CNTF (0.4 µg) or bFGF (1 µg) after LIP were examined on wholemounts at 7 days. SD-OCT showed damage in a circular region of the superotemporal retina, whose diameter varied from 1,842.4±84.5 µm (at 24 hours) to 1,407.7±52.8 µm (at 7 days). This region had a progressive thickness diminution from 183.4±5 µm (at 12 h) to 114.6±6 µm (at 7 d). Oriented cross-sections showed within the light-damaged region of the retina massive loss of rods and cone-photoreceptors. Wholemounts documented a circular region containing lower numbers of L- and S-cones. Within a circular area (1 mm or 1.3 mm radius, respectively) in the left and in its corresponding region of the contralateral-fellow-retina, total L- or S-cones were 7,118±842 or 661±125 for the LED exposed retinas (n = 7) and 14,040±1,860 or 2,255±193 for the fellow retinas (n = 7), respectively. BMD, BDNF, PEDF and bFGF but not CNTF showed significant neuroprotective effects on L- or S-cones. We conclude that LIP results in rod and cone-photoreceptor loss, and is a reliable, quantifiable model to study cone-photoreceptor degeneration. Intravitreal BDNF, PEDF or bFGF, or topical BMD afford significant cone neuroprotection in this model.

Retinal neurodegeneration in experimental glaucoma

In rats and mice, limbar tissues of the left eye were laser-photocoagulated (LP) and ocular hypertension (OHT) effects were investigated 1 week to 6 months later. To investigate the innermost layers, retinas were examined in wholemounts using tracing from the superior colliculi to identify retinal ganglion cells (RGCs) with intact retrograde axonal transport, melanopsin immunodetection to identify intrinsically photosensitive RGCs (m(+)RGC), Brn3a immunodetection to identify most RGCs but not m(+)RGCs, RECA1 immunodetection to examine the inner retinal vessels, and DAPI staining to detect all nuclei in the GC layer. The outer retinal layers (ORLs) were examined in cross sections analyzed morphometrically or in wholemounts to study S- and L-cones. Innervation of the superior colliculi was examined 10 days to 14 weeks after LP with orthogradely transported cholera toxin subunit B. By 2 weeks, OHT resulted in pie-shaped sectors devoid of FG(+)RGCs or Brn3a(+)RGCs but with large numbers of DAPI(+)nuclei. Brn3a(+)RGCs were significantly greater than FG(+)RGCs, indicating the survival of large numbers of RGCs with their axonal transport impaired. The inner retinal vasculature showed no abnormalities that could account for the sectorial loss of RGCs. m(+)RGCs decreased to approximately 50-51% in a diffuse loss across the retina. Cross sections showed focal areas of degeneration in the ORLs. RGC loss at 1m diminished to 20-25% and did not progress further with time, whereas the S- and L-cone populations diminished progressively up to 6m. The retinotectal projection was reduced by 10 days and did not progress further. LP-induced OHT results in retrograde degeneration of RGCs and m(+)RGCs, severe damage to the ORL, and loss of retinotectal terminals.