Scotopic Threshold Response Research Articles

Comparison of electroretinographic responses between two different age groups of adult Dark Agouti rats.

To describe and compare the differences in electroretinographic responses between two different age groups of adult Dark Agouti (DA) rats and to better understand the effect of age on retinal histology and function. The electroretinographic responses of two different age groups of adult DA rats were compared. Animals were divided into younger adult DA rats 10-12wk (n=8) and older adult DA rats 17-19wk (n=8). Full field electroretinography (ERG) was recorded simultaneously from both eyes after dark adaption and light adaption and parameters including the positive scotopic threshold response (pSTR), negative scotopic threshold response (nSTR), scotopic a-wave, b-wave, photopic a-wave, b-wave and photopic negative response (PhNR) were compared between groups. The older adult rats displayed lower stimulation thresholds of the STRs (pSTR and nSTR) and higher amplitudes of pSTR, scotopic a-wave and b-wave, photopic b-wave and PhNR amplitudes, with shorter implicit times. Photopic a-wave amplitudes were however higher in the younger adult rats. In summary, for the rod system, photoreceptor, bipolar cell and RGC activity was enhanced in the older adult rats. For the cone system, RGC and bipolar cell activity was enhanced, while photoreceptor activity was depressed in the older adult rats. Such age-related selective modification of retinal cell function needs to be considered when conducting ophthalmic research in adult rats.

Caspase-7: a critical mediator of optic nerve injury-induced retinal ganglion cell death.

BackgroundAxonal injury of the optic nerve (ON) is involved in various ocular diseases, such as glaucoma and traumatic optic neuropathy, which leads to apoptotic death of retinal ganglion cells (RGCs) and loss of vision. Caspases have been implicated in RGC pathogenesis. However, the role of caspase-7, a functionally unique caspase, in ON injury and RGC apoptosis has not been reported previously. The purpose of this study is to evaluate the role of caspase-7 in ON injury-induced RGC apoptosis.ResultsC57BL/6 (wildtype, WT) and caspase-7 knockout (Casp7−/−) mice were used. We show that ON crush activated caspase-7 and calpain-1, an upstream activator of caspase-7, in mouse RGCs, as well as hydrolysis of kinectin and co-chaperone P23, specific substrates of caspase-7. ON crush caused a progressive loss of RGCs to 28 days after injury. Knockout of caspase-7 partially and significantly protected against the ON injury-induced RGC loss; RGC density at 28 days post ON crush in Casp7−/− mice was approximately twice of that in WT ON injured retinas. Consistent with changes in RGC counts, spectral-domain optical coherence tomography analysis revealed that ON crush significantly reduced the in vivo thickness of the ganglion cell complex layer (including ganglion cell layer, nerve fiber layer, and inner plexiform layer) in the retina. The ON crush-induced thinning of retinal layer was significantly ameliorated in Casp7−/− mice when compared to WT mice. Moreover, electroretinography analysis demonstrated a decline in the positive component of scotopic threshold response amplitude in ON crushed eyes of the WT mice, whereas this RGC functional response was significantly higher in Casp7−/− mice at 28 days post injury.ConclusionAltogether, our findings indicate that caspase-7 plays a critical role in ON injury-induced RGC death, and inhibition of caspase-7 activity may be a novel therapeutic strategy for glaucoma and other neurodegenerative diseases of the retina.Electronic supplementary materialThe online version of this article (doi:10.1186/s13024-015-0039-2) contains supplementary material, which is available to authorized users.

The effect of intraocular and intracranial pressure on retinal structure and function in rats.

An increasing number of studies indicate that the optic nerve head of the eye is sensitive not only to changes in intraocular pressure (IOP), but also to intracranial pressure (ICP). This study examines changes to optic nerve and retinal structure in a rat model in response to a range of IOP and ICP levels using optical coherence tomography. Furthermore, we examine the functional sequelae of these structural changes by quantifying the effect of pressure changes on the electroretinogram. IOP elevation (10–90 mmHg) induces progressive deformation of the optic nerve head and retinal surface (P < 0.05), compression of the retina (P < 0.05) and bipolar cell (b-wave), and retinal ganglion cell (scotopic threshold response) dysfunction (P < 0.05). Simultaneously altering ICP (−5 to 30 mmHg) modifies these IOP-induced responses, with lower ICP (−5 mmHg) exacerbating and higher ICP (15–30 mmHg) ameliorating structural and functional deficits. Thus, the balance between IOP and ICP (optic nerve pressure gradient, ONPG = IOP − ICP) plays an important role in optic nerve integrity. Structural and functional parameters exhibit a two-phase relationship to ONPG, with structural changes being more sensitive to ONPG modification (threshold = −0.6 to 11.3 mmHg) compared with functional changes (threshold = 49.7–54.6 mmHg). These findings have implications for diseases including glaucoma, intracranial hypertension, and long-term exposure to microgravity.

In vivo electroretinographic studies of the role of GABAC receptors in retinal signal processing

All three classes of receptors for the inhibitory neurotransmitter GABA (GABAR) are expressed in the retina. This study investigated roles of GABAR, especially GABACR (GABA(A)-ρ), in retinal signaling in vivo by studying effects on the mouse electroretinogram (ERG) of genetic deletion of GABACR versus pharmacological blockade using receptor antagonists. Brief full-field flash ERGs were recorded from anesthetized GABACR−/− mice, and WT C57BL/6 (B6) mice, before and after intravitreal injection of GABACR antagonists, TPMPA, 3-APMPA, or the more recently developed 2-AEMP; GABAAR antagonist, SR95531; GABABR antagonist, CGP, and agonist, baclofen. Intravitreal injections of TPMPA and SR95531 were also made in Brown Norway rats. The effect of 2-AEMP on GABA-induced current was tested directly in isolated rat rod bipolar cells, and 2-AEMP was found to preferentially block GABACR in those cells. Maximum amplitudes of dark (DA) and light-adapted (LA) ERG b-waves were reduced in GABACR−/− mice, compared to B6 mice, by 30–60%; a-waves were unaltered and oscillatory potential amplitudes were increased. In B6 mice, after injection of TPMPA (also in rats), 3-APMPA or 2-AEMP, ERGs became similar to ERGs of GABACR−/− mice. Blockade of GABAARs and GABABRs, or agonism of GABABRs did not alter B6 DA b-wave amplitude. The negative scotopic threshold response (nSTR) was slightly less sensitive in GABACR−/− than in B6 mice, and unaltered by 2-AEMP. However, amplitudes of nSTR and photopic negative response (PhNR), both of which originate from inner retina, were enhanced by TPMPA and 3-APMPA, each of which has GABAB agonist properties, and further increased by baclofen. The finding that genetic deletion of GABACR, the GABACR antagonist 2-AEMP, and other antagonists all reduced ERG b-wave amplitude, supports a role for GABACR in determining the maximum response amplitude of bipolar cells contributing to the b-wave. GABACR antagonists differed in their effects on nSTR and PhNR; antagonists with GABAB agonist properties enhanced light-driven responses whereas 2-AEMP did not.

Chronic ocular hypertension induced by circumlimbal suture in rats.

To induce chronic intraocular pressure (IOP) elevation in rat eyes by circumlimbal suture. Anesthetized (isoflurane) Long-Evans rats underwent unilateral circumlimbal suture implantation while the fellow eyes served as untreated controls (n = 15). A sham group (n = 8) received the same procedure except that the suture was loosely tied. Intraocular pressure, electroretinography (ERG), and optical coherence tomography (OCT) were monitored for 15 weeks, after which retinal histology and immunofluorescence staining for glial fibrillary acidic protein (GFAP) and ionized calcium binding adapter molecule-1 (Iba-1) were undertaken. Both IOP and ERG remained unaltered in the sham and all control eyes over 15 weeks. In the ocular hypertensive eye, IOP spiked from 17 ± 1 to 58 ± 3 mm Hg immediately after suture application, recovering to 32 ± 2 mm Hg by 24 hours, and remained elevated by 7 to 10 mm Hg above baseline for 15 weeks. At week 2, there was a small reduction of ERG components involving the photoreceptor a-wave, bipolar cell b-wave, and ganglion cell-mediated scotopic threshold response (pSTR). The reduction in a- and b-wave remained stable, while the pSTR became more affected from week 8 onward (P < 0.05). By week 12, there was progressive retinal nerve fiber layer (RNFL) thinning. At week 15, GFAP expression was upregulated in inner retina and on Müller cells. The ganglion cell dysfunction was associated with RNFL thinning and cell loss in the ganglion cell layer. Circumlimbal suture provides a simple and cost-effective way to induce mild chronic ocular hypertension in rat eyes. This model produces preferential ganglion cell dysfunction and RNFL reduction.

Retinal Ganglion Cell Loss and Mild Vasculopathy in Methylene Tetrahydrofolate Reductase (Mthfr)-Deficient Mice: A Model of Mild Hyperhomocysteinemia.

Methylenetetrahydrofolate reductase (Mthfr) is a key enzyme in homocysteine-methionine metabolism. We investigated Mthfr expression in retina and asked whether mild hyperhomocysteinemia, due to Mthfr deficiency, alters retinal neurovascular structure and function. Expression of Mthfr was investigated at the gene and protein level using quantitative (q) RT-PCR, in situ hybridization, immunoblotting, and immunohistochemistry (IHC). The Mthfr+/+ and Mthfr+/- mice were subjected to comprehensive evaluation using ERG, funduscopy, fluorescein angiography (FA), spectral-domain optical coherence tomography (SD-OCT), HPLC, and morphometric and IHC analysis of glial fibrillary acidic protein (GFAP) at 8 to 24 weeks. Gene and protein analyses disclosed widespread retinal expression of Mthfr. Electroretinography (ERG) revealed a significant decrease in positive scotopic threshold response in retinas of Mthfr+/- mice at 24 weeks. Fundus examination in mice from both groups was normal; FA revealed areas of focal vascular leakage in 20% of Mthfr+/- mice at 12 to 16 weeks and 60% by 24 weeks. The SD-OCT revealed a significant decrease in nerve fiber layer (NFL) thickness at 24 weeks in Mthfr+/- compared to Mthfr+/+ mice. There was a 2-fold elevation in retinal hcy at 24 weeks in Mthfr+/- mice by HPLC and IHC. Morphometric analysis revealed an approximately 20% reduction in cells in the ganglion cell layer of Mthfr+/- mice at 24 weeks. The IHC indicated significantly increased GFAP labeling suggestive of Müller cell activation. Mildly hyperhomocysteinemic Mthfr+/- mice demonstrate reduced ganglion cell function, thinner NFL, and mild vasculopathy by 24 weeks. The retinal phenotype is similar to that of hyperhomocysteinemic mice with deficiency of cystathionine-β-synthase (Cbs) reported earlier. The data support the hypothesis that hyperhomocysteinemia may be causative in certain retinal neurovasculopathies.

Molecular, anatomical and functional changes in the retinal ganglion cells after optic nerve crush in mice.

Optic nerve crush (ONC) and subsequent axonal damage can be used in rodents to study the mechanism of retinal ganglion cell (RGC) degeneration. Here, we examined electroretinograms (ERGs) in post-ONC mice to investigate changes in the positive scotopic threshold response (pSTR). We then compared these changes with molecular and morphological changes to identify early objective biomarkers of RGC dysfunction. Fifty 12-week-old C57BL/6 mice were included. ONC was used to induce axonal injury in the right eye of each animal, with the left eye used as a control. The expression of the RGC markers Brn3a and Brn3b was measured on days 1, 2, 3, 5 and 7 after ONC with quantitative real-time PCR. ERGs were recorded under dark adaptation with the stimulus intensity increasing from -6.2 to 0.43 log cd-s/m(2) on days 1, 2, 3, 5, 7 and 10 after ONC. The pSTR, a- and b-wave amplitudes were measured. Inner retinal thickness around the optic nerve head was measured with spectral-domain optical coherence tomography on days 0, 2, 5, 7 and 10 after ONC. The expression of Brn3a and Brn3b began to significantly decrease on day 1 and day 2, respectively (P<0.01). The amplitude of the pSTR underwent rapid, significant deterioration on day 3, after which it fell gradually (P<0.01), while the a- and b-wave amplitudes remained unchanged throughout the experiment. Inner retinal thickness gradually decreased, with the most significant reduction on day 10 (P<0.01). Decrease in pSTR likelyreflected the early loss of RGC function after ONC and that declining expression of RGC-specific genes preceded anatomical and functional changes in the RGCs.

Chronic intraocular pressure elevation impairs autoregulatory capacity in streptozotocin-induced diabetic rat retina.

To assess ocular blood flow responses to acute IOP stress following 4weeks of chronic IOP elevation in streptozotocin (STZ)-induced diabetic and control rats. We hypothesise that chronic IOP elevation for 4weeks will further impair blood flow regulation in STZ-induced diabetic rats eyes. Two weeks following citrate buffer or STZ-injections chronic IOP elevation was induced in Long Evans rats via fortnightly intracameral injections of microspheres (15μm) suspended in 5% polyethylene glycol. IOP was monitored daily. Electroretinography (ERG, -6.79-2.07 log cdsm(-2) ) was undertaken at Week 4 to compare photoreceptor (RmPIII ), ON-bipolar cell (Vmax ) and ganglion cell dominant ERG [scotopic threshold response (STR)] components. 4weeks post-chronic IOP induction, ocular blood flow (laser Doppler flowmetry) was measured in response to acute IOP challenge (10-100mmHg, in 5mmHg steps, each 3min). Four weeks of chronic IOP (mean±S.E.M., citrate: 24.0±0.3 to 30.7±1.3 and STZ-diabetes: 24.2±0.2 to 31.1±1.2mmHg) was associated with reduced photoreceptor amplitude in both groups (-25.3±2.2% and -17.2±3.0%, respectively). STZ-diabetic eyes showed reduced photoreceptor sensitivity (citrate: 0.5±1.8%, STZ-diabetic: -8.1±2.4%). Paradoxically ON-bipolar cell sensitivity was increased, particularly in citrate control eyes (citrate: 166.8±25.9%, STZ-diabetic: 64.8±18.7%). The ganglion cell dominant STR was not significantly reduced in STZ-diabetic rats. Using acute IOP elevation to probe autoregulation, we show that STZ-diabetes impaired autoregulation compared with citrate control animals. The combination of STZ-diabetes and chronic IOP elevation further impaired autoregulation. STZ-diabetes and chronic IOP elevation appear to be additive risk factors for impairment of ocular blood flow autoregulation.

Role of C/EBP homologous protein in retinal ganglion cell death after ischemia/reperfusion injury.

To investigate the role of C/EBP homologous protein (CHOP), a proapoptotic protein, and the unfolded protein response (UPR) marker that is involved in endoplasmic reticulum (ER) stress-mediated apoptosis in mouse retinal ganglion cell (RGC) death following ischemia/reperfusion (I/R) injury. Retinal I/R injury was induced in adult C57BL/6J wild-type (WT) and CHOP knockout (Chop(-/-)) mice by raising IOP to 120 mm Hg for 60 minutes. Expression of CHOP and other UPR markers was studied by Western blot and immunohistochemistry. Retinal ganglion cell counts were performed in retinal flat mounts stained with an RGC marker. Retinal ganglion cell function was evaluated by scotopic threshold response (STR) electroretinography. In WT mice, retinal CHOP was upregulated by 30% in I/R-injured eyes compared to uninjured eyes 3 days after injury (P < 0.05). Immunohistochemistry confirmed CHOP upregulation specifically in RGCs. CHOP knockout did not affect baseline RGC density or STR amplitude. Ischemia/reperfusion injury decreased RGC densities and STR amplitudes in both WT and Chop(-/-) mice. However, survival of RGCs in I/R-injured Chop(-/-) mouse was 48% higher (P < 0.05) than that in I/R-injured WT mouse 3 days after I/R injury. Similarly, RGC density was significantly higher in Chop(-/-) eyes at 7, 14, and 28 days after I/R injury. Scotopic threshold response amplitudes of Chop(-/-) mice were significantly higher at 3 and 7 days after I/R than those of WT mice. Absence of CHOP partially protects against RGC loss and reduction in retinal function after I/R injury, indicating that CHOP and, thus, ER stress play an important role in RGC apoptosis in retinal I/R injury.

Prolonged elevation of intraocular pressure results in retinal ganglion cell loss and abnormal retinal function in mice

The purpose of this study was to assess the impact of prolonged intraocular pressure (IOP) elevation on retinal anatomy and function in a mouse model of experimental glaucoma. IOP was elevated by anterior chamber injection of a fixed combination of polystyrene beads and sodium hyaluronate, and maintained via re-injection after 24 weeks. IOP was measured weekly with a rebound tonometer for 48 weeks. Histology was assessed with a combination of retrograde labeling and antibody staining. Retinal physiology and function was assessed with dark-adapted electroretinograms (ERGs). Comparisons between bead-injected animals and various controls were conducted at both 24 and 48 weeks after bead injection. IOP was elevated throughout the study. IOP elevation resulted in a reduction of retinal ganglion cell (RGCs) and an increase in axial length at both 24 and 48 weeks after bead injection. The b-wave amplitude of the ERG was increased to the same degree in bead-injected eyes at both time points, similar to previous studies. The positive scotopic threshold response (pSTR) amplitude, a measure of RGC electrical function, was diminished at both 24 and 48 weeks when normalized to the increased b-wave amplitude. At 48 weeks, the pSTR amplitude was reduced even without normalization, suggesting more profound RGC dysfunction. We conclude that injection of polystyrene beads and sodium hyaluronate causes chronic IOP elevation which results in phenotypes of stable b-wave amplitude increase and progressive pSTR amplitude reduction, as well as RGC loss and axial length elongation.

Examination of VLC-PUFA-deficient photoreceptor terminals.

Juvenile-onset autosomal dominant Stargardt-like macular dystrophy (STGD3) is caused by mutations in ELOVL4 (elongation of very long fatty acids-4), an elongase necessary for the biosynthesis of very long chain fatty acids (VLC-FAs ≥ C26). Photoreceptors are enriched with VLC polyunsaturated fatty acids (VLC-PUFAs), which are necessary for long-term survival of rod photoreceptors. The purpose of these studies was to determine the effect of deletion of VLC-PUFAs on rod synaptic function in retinas of mice conditionally depleted (KO) of Elovl4. Retina function was assessed in wild-type (WT) and KO by electroretinography. Outer plexiform structure was evaluated by immunofluorescence and transmission electron microscopy. Single-cell recordings measured rod ion channel operation and rod bipolar glutamate signaling. Sucrose gradient centrifugation was used to isolate synaptosomes from bovine retina. Proteins and lipids were analyzed by Western blotting and tandem mass spectroscopy, respectively. Inner retinal responses (b-wave, oscillatory potentials, and scotopic threshold responses) of the ERG were decreased in the KO mice compared to controls. However the rod ion channel operation and bipolar glutamate responses were comparable between groups. Biochemical analysis revealed that conventional and ribbon synapses have VLC-PUFAs. Ultrastructural analysis showed that the outer plexiform layer was disorganized and the diameter of vesicles in rod terminals was smaller in the KO mice. Very long chain PUFAs affect rod function by contributing to synaptic vesicle size, which may alter the dynamics of synaptic transmission, ultimately resulting in a loss of neuronal connectivity and death of rod photoreceptors.

Monitoring retinal morphologic and functional changes in mice following optic nerve crush.

We characterized the morphologic and functional changes in optic nerve crushed mice and evaluated electroretinogram (ERG) responses as tools to monitor retinal ganglion cell (RGC) dysfunction. We performed optic nerve crush (ONC) unilaterally in adult BALB/cJ mice. The neuronal loss in the RGC layer (GCL) and superior colliculus (SC) was determined by Nissl staining. Retinal thickness was assessed by spectral-domain optical coherence tomography (SD-OCT) imaging. Retinal function was determined by pattern ERG and full-field flash ERG. Responses of pattern ERG, positive scotopic threshold response (pSTR), scotopic oscillatory potentials (OPs), and photopic negative response (PhNR) were analyzed. The ONC induced progressive neuronal loss in GCL and contralateral SC starting from 7 and 28 days following ONC, respectively. A linear correlation was observed between combined thickness of nerve fiber layer (NFL), GCL, and inner plexiform layer (IPL) imaged by SD-OCT and cell numbers in GCL. Only half of the normal BALB/cJ mice exhibited pattern ERG response, which was smaller and later compared to C57BL/6J mice. The ONC reduced pattern ERG and pSTR, but the reduction of pattern ERG was earlier than pSTR, preceding the anatomical cell loss in the GCL. The PhNR and scotopic OPs were not affected by ONC. The SD-OCT and ERG can be used to monitor noninvasively retinal morphologic and functional changes induced by ONC. Pattern ERG and pSTR are able to detect early RGC dysfunction, but pattern ERG exhibits higher sensitivity. Our results support the use of these tools in studies using the mouse ONC model.

FTY720 protects retinal ganglion cells in experimental glaucoma.

To investigate the neuroprotective effects of sphingosine-1-phosphate (S1P) analogue fingolimod (FTY720) in experimental glaucoma in rats. A unilateral chronic ocular hypertensive model was established by injections of microbeads into the anterior eye chamber of adult Sprague-Dawley rats. Fingolimod was administered to one group of rats intraperitoneally every week for 3 months. The scotopic threshold response (STR) was recorded to assess the function of the inner retina. Changes in cell density in the ganglion cell layer (GCL) were evaluated by hematoxylin and eosin staining on retinal sections and axonal count of the optic nerve was performed using Bielschowsky's silver staining. Effects of drug treatment on activation of Akt and Erk1/2 were evaluated using Western blotting by assessing phosphorylation levels of these proteins. The expression of S1P receptors in the optic nerve head region was also evaluated using Western blotting and immunohistochemistry. Administration of FTY720 reduced the loss of STR amplitude in glaucomatous eyes (P < 0.05). Counting and plotting the cell numbers/axonal density showed significant neural preservation in the GCL and the optic nerve (P < 0.05). An increased phosphorylation level of Akt and Erk1/2 following FTY720 administration was observed. Both S1P1 and S1P5 receptors were found to be expressed in the retina and the expression of S1P1R was upregulated in experimentally-induced glaucoma. This study demonstrates, for the first time, that FTY720 could act as a neuroprotective agent to protect retinal ganglion cells in experimental glaucoma. Administration of this drug significantly reduces the structural and functional loss of the inner retina elicited indicating that it may potentially be used to attenuate neuronal loss and optic nerve damage in glaucomatous patients.

Contribution of retinal ganglion cells to the mouse electroretinogram

To quantify the direct contribution of retinal ganglion cells (RGCs) on individual components of the mouse electroretinogram (ERG). Dark- and light-adapted ERGs from mice 8 to 12 weeks after optic nerve transection (ONTx, n=14) were analyzed through stimulus response curves for a- and b-waves, oscillatory potentials (OPs), positive and negative scotopic threshold response (p/n STR), and the photopic negative response (PhNR) and compared with unoperated and sham-operated controls, as well as to eyes treated with 6-cyano-7-nitroquinoxaline-2,3-dion (CNQX). We confirmed in mice that CNQX intravitreal injection reduced the scotopic a-wave amplitude at high flash strength, confirming a post-receptoral contribution to the a-wave. We found that ONTx, which is more specific to RGCs, did not affect the a-wave amplitude and implicit time in either photopic or scotopic conditions while the b-wave was reduced. Both the pSTR and nSTR components were reduced in amplitude, with the balance between the two components resulting in a shortening of the nSTR peak implicit time. On the other hand, amplitude of the PhNR was increased while the OPs were minimally affected. With an intact a-wave demonstrated following ONTx, we find that the most robust indicators of RGC function in the mouse full-field ERG were the STR components.

Assessment of inner retina dysfunction and progressive ganglion cell loss in a mouse model of glaucoma

The DBA/2J mouse is a model of ocular hypertension and retinal ganglion cell (RGC) degeneration, the main features of which are iris pigment dispersion (IPD) and iris stromal atrophy (ISA). These animals also experience glaucomatous changes, including an increase in intraocular pressure (IOP) beginning at about 9–12 months of age and sectorial RGC death in the retina. The aim of this study was to determine the onset of functional changes exhibited by DBA/2J mice in the inner retina. This was performed by means of electroretinographic recordings (scotopic threshold response, STR) and their correlation with morphological changes (loss of RGCs). To this end, we recorded the scotopic threshold response in control C57BL/6J and in DBA/2J mice at different ages. The RGCs, in both DBA/2J and C57BL/6J animals, were identified at 15 months of age by retrograde tracing with an analogue of fluorogold, hydroxystilbamidine methanesulfonate (OHSt), applied on the superior colliculi. Whole mount retinas were processed to quantify the population of RGCs identified by fluorogold tracing and Brn3a immunodetection, and were counted using image analysis software; an isodensity contour plot was generated for each retina. DBA/2J mice showed a significant reduction in the positive STR (pSTR) amplitudes at 12 months of age, as compared to control C57BL/6J mice of the same age. The pSTR mean amplitude decreased to approximately 27.82% of the values recorded in control mice (p = 0.0058). STR responses decreased in both strains as a result of the natural process of aging, but the decrease was more pronounced in DBA/2J mice. Furthermore, quantification of the total number of RGCs identified by OHSt and Brn3a expression showed a reduced population of RGCs in DBA/2J mice as compared to control mice. Regression analysis revealed significant correlations between the decrease in pSTR and a non-homogeneous reduction in the number of RGCs throughout the retina. Our results indicate the existence of a correlation between retinal function impairment and RGC loss. This functional and morphological analysis allows a reliable assessment of the progression of the disease.

The Photopic Negative Response of the Mouse Electroretinogram: Reduction by Acute Elevation of Intraocular Pressure

To determine the presence and magnitude of the photopic negative response (PhNR) component of the electroretinogram (ERG) in the mouse eye and to test if it is altered by short-term elevation of intraocular pressure (IOP). Photopic and scotopic ERGs were recorded from 12-month-old C57BL/6J mice and analyzed for photoreceptoral responses (a-wave), bipolar cell responses (b-wave), scotopic threshold responses (STRs), and PhNRs. Electroretinogram signals were measured before and after short-term subischemic elevation of IOP (50 mm Hg for 30 minutes) induced by cannulation of the anterior chamber. Retinas were subsequently assessed for signs of retinal stress and cell survival using immunohistochemistry and quantitative PCR. The corneal negative PhNR of the photopic ERG was elicited in the mouse eye, and its amplitudes correlated with amplitudes of the positive STR (pSTR). Elevation of IOP significantly reduced amplitudes of both the PhNR and pSTR, while scotopic a-waves, scotopic b-waves, and photopic b-waves were unchanged. Pressure elevation was associated with upregulation of glial fibrillary acidic protein and heme oxygenase 1 expression in retinal macroglia in the absence of retinal cell death. The PhNR component of the full-field ERG can be recorded in mice and is sensitive to elevation of IOP. Correlation between PhNR and pSTR signals before and after IOP elevation suggests that the PhNR depends on inner retinal integrity and provides a means for evaluating inner retinal function in mouse models.

Effects of l-arginine on anatomical and electrophysiological deterioration of the eye in a rodent model of nonarteritic ischemic optic neuropathy

The aims of this study were to clarify the effectiveness of L-arginine (1) for reducing the severity of anatomical changes in the eye and improving visual function in the acute stage of a rodent model of nonarteritic ischemic optic neuropathy (rNAION) and (2) in preventing those changes in anatomy and visual function. For the first aim, L-arginine was intravenously injected into rats 3 h after rNAION induction; for the second aim, rNAION was induced after the oral administration of L-arginine for 7 days. The inner retinal thickness was determined over time by optical coherence tomography, and the amplitude of the scotopic threshold response (STR) and the number of surviving retinal ganglion cells (RGCs) were measured. These data were compared with the baseline data from the control group. Both intravenous infusion of L-arginine after rNAION induction and oral pretreatment with L-arginine significantly decreased optic disc edema in the acute stage and thinning of the inner retina, reduced the decrease in STR amplitude, and reduced the decrease in the number of RGCs during rNAION. Based on these results, we conclude that L-arginine treatment is effective for reducing anatomical changes in the eye and improving visual function in the acute stage of rNAION and that pretreatment with L-arginine is an effective therapy to reduce the severity of the condition during recurrence in the other eye.

Effects of steroid administration and transcorneal electrical stimulation on the anatomic and electrophysiologic deterioration of nonarteritic ischemic optic neuropathy in a rodent model

To elucidate the effectiveness of steroid administration and transcorneal electrical stimulation (TES) on anatomic changes and visual function in a rodent model of nonarteritic ischemic optic neuropathy (rNAION). Methylprednisolone (20 mg/kg) was injected through a central venous catheter twice a day for 3 days. TES was delivered with biphasic square pulses of 1 ms/phase, 100 μA of current, and 20 Hz of frequency for 60 min 3 h after induction on the 1st, 4th, 7th, 14th, and 28th days. Intravenous infusion of methylprednisolone significantly decreased the degree of acute disc edema but did not preserve the inner retinal thinning, decreasing the amplitude of scotopic threshold responses (STR) and decreasing retinal ganglion cell (RGC) numbers in rNAION. TES preserved the decreasing STR amplitude and the decreasing RGC numbers in rNAION. Steroids are effective for reducing disc edema in the acute stage in rNAION. TES is effective for preserving decreasing RGC numbers and function in the chronic stage of rNAION.

Elevated Intraocular Pressure Causes Inner Retinal Dysfunction Before Cell Loss in a Mouse Model of Experimental Glaucoma

We assessed the relationship among intraocular pressure (IOP), histology, and retinal function changes in a mouse model of induced, chronic, mild ocular hypertension. IOP was elevated experimentally via anterior chamber injection of polystyrene beads and measured twice weekly with a rebound tonometer. Histology was assessed with a combination of neurobiotin (NB) retrograde labeling of retinal ganglion cells (RGCs) and TO-PRO3 staining. Retinal function was assessed with serial dark-adapted electroretinograms (ERGs) optimized for detection of the a-wave, b-wave, and positive and negative scotopic threshold responses (pSTR, nSTR). Comparisons between bead-injected and saline-injected (control) eyes were conducted. IOP remained elevated for at least 3 months following a single injection of polystyrene beads. Elevated IOP resulted in a mild, progressive reduction of RGCs, and a mild increase in axial length at 6 and 12 weeks after bead injection. The raw b-wave amplitude was increased shortly after IOP elevation, but the raw a-wave, pSTR, and nSTR amplitudes were unchanged. pSTR and nSTR amplitudes were normalized to the increased b-wave. With this normalization, the pSTR amplitude was decreased shortly after IOP elevation. Polystyrene bead injection results in a mild, chronic elevation of IOP that recapitulates several critical aspects of human ocular hypertension and glaucoma, and results in early changes in retinal electrical function that precede histologic changes. It is possible that glaucoma associated with elevated IOP involves the early disruption of a complex combination of retinal synapses.

Electroretinographical and histological study of mouse retina after optic nerve section: a comparison between wild‐type and retinal degeneration 1 mice

Retinal ganglion cell death underlies the pathophysiology of neurodegenerative disorders such as glaucoma or optic nerve trauma. To assess the potential influence of photoreceptor degeneration on retinal ganglion cell survival, and to evaluate functionality, we took advantage of the optic nerve section mouse model. Surviving retinal ganglion cells were double-stained by exposing both superior colliculi to fluorogold, and by applying dextran-tetramethylrhodamine to the injured optic nerve stump. To assess retinal function in wild-type animals, electroretinograms were recorded on the injured eyes and compared with the contralateral. Similar labelling experiments were carried out on retinal degeneration 1 mice. Surviving retinal ganglion cells were counted 21 days after axotomy and compared with wild-type mice. No functional experiments were performed on retinal degeneration 1 animals because they do not develop normal electroretinographical responses. A significant decrease in retinal ganglion cell density was observed 6 days after axotomy in the wild type. Functional studies revealed that, in scotopic conditions, axotomy induced a significant amplitude decrease in the positive scotopic threshold response component of the electroretinogram. Such decrease paralleled cell loss, suggesting it may be an appropriate technique to evaluate functionality. When comparing retinal ganglion cell densities in wild-type and retinal degeneration 1 mice, a significant greater survival was observed on the latter. After optic nerve section, electroretinographical recordings exhibited a progressive decrease in the amplitude of the positive scotopic threshold response wave, reflecting ganglion cell loss. Interestingly, rod degeneration seemed, at least initially, to protect from axotomy-driven damage.