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. Author manuscript; available in PMC: 2014 Aug 27.
Published in final edited form as: Adv Exp Med Biol. 2012;723:495–502. doi: 10.1007/978-1-4614-0631-0_62

Rod Photoreceptor Temporal Properties in Retinal Degenerative Diseases

Yuquan Wen 1,✉, Kirsten G Locke 2, Donald C Hood 3, David G Birch 4,5
PMCID: PMC4145877  NIHMSID: NIHMS617289  PMID: 22183369

62.1 Introduction

Among the many genetic causes of retinal degenerative disease (RDD), some predominantly affect the rods, while others predominantly affect the cones (RetNet, http://www.sph.uth.tmc.edu/RetNet/disease.htm). Because rhodopsin, for example, is unique to the rods (Liebman et al. 1974), rhodopsin mutations cause the characteristic early night blindness typical of retinitis pigmentosa (RP). Initial loss of rods is followed by secondary cone degeneration as the disease progresses (Petters et al. 1997; Koenekoop 2009; Punzo et al. 2009).

In contrast to RP, patients with cone–rod dystrophy (CRD) initially exhibit dysfunction of the cone photoreceptors and rod-mediated losses are minimal in the early stages. However, in later stage of CRD, rods also degenerate, causing elevated dark-adapted thresholds and reduction in the amplitude of dark-adapted rod electroretinogram (ERG) (Fishman 1976). Stargardt disease represents a variant of CRD, with dystrophy limited primarily to the posterior pole. It is predominantly an autosomal recessive macular dystrophy caused by ABCA4 (ATP-binding cassette, subfamily A, member 4) mutations (Allikmets et al. 1997a, b; Fishman 2010).

It is unknown whether rod photoresponse kinetics are altered in these human RDDs, although studies in laboratory models of RP showed earlier-than-normal rod photoresponse recovery (Niculescu 2004; Kraft et al. 2005; Wen et al. 2006; Wen 2008; Wen and Kraft 2008). Here, we use paired-flash full-field ERG to characterize the rod photoresponse recovery kinetics in patients with primary rod degeneration (RP), patients with secondary rod degeneration (CRD), and patients with degeneration limited to the posterior pole (Stargardt disease).

62.2 Materials and Methods

62.2.1 Subjects

Patients were recruited from the database of the Southwest Eye Registry at the Retina Foundation of the Southwest. They included 18 patients with autosomal dominant retinitis pigmentosa (adRP) reported previously (Wen et al. 2011), 5 patients with CRD, and 4 patients with Stargardt disease. The 18 adRP patients included 10 patients harboring rhodopsin mutations and 8 patients harboring peripherin/rds mutations. Included within the 5 patients diagnosed with CRD were two autosomal recessive/isolate CRD patients heterozygous for an ABCA4 mutation (ABCA4: Asn-965-Ser or Ala-192-Thr) and one autosomal dominant patient carrying a peripherin/rds mutation (Arg-172-Trp). Individuals with normal eye exams (n = 13) provided normative values.

In order to ensure reliable analysis of pair-flash ERG results, patients were recruited only if they retained a dark-adapted rod response amplitude of greater than 10 μV to a ISCEV (International Society for Clinical Electrophysiology of Vision) standard rod stimulus. The tenets of the Declaration of Helsinki were followed and all subjects gave written informed consent after a full explanation of the procedures was given. All procedures were approved by the institutional review board of University of Texas Southwestern Medical Center, Dallas, TX.

62.2.2 Evaluation of the Rod Inactivation Kinetics Using Paired-Flash ERG

Prior to paired-flash ERG, the pupil of the left eye was dilated (1% tropicamide and 2.5% phenylephrine hydrochloride), and the patient was dark adapted for 45 min. Paired-flash ERG was recorded in a ganzfeld dome. A bipolar contact lens (Burian-Allen electrode, Hansen Laboratories, Coralville, IA) was used to record the signals. Details of pair-flash ERG recording were previously described (Birch et al. 1995; Pepperberg et al. 1997). Briefly, a test flash (achromatic, 2.4 log scot td-s) was delivered prior to the probe flash (λcut-off = 470 nm, 4.2 log scot td-s) with variable inter-stimulus-interval (ISI). The cone response at short (i.e., 200 ms) ISIs was subtracted from the response to the probe flash to provide a rod-only response at each ISI.

62.3 Results

62.3.1 Rod Photoresponse Recovery Kinetics in RDDs

To determine whether rod photoresponse recovery kinetics in patients with RDDs is different from normal, we derived Tsat from these patients using the paired-flash paradigm. Figure 62.1a–d shows the rod-only response to probe flash at indicated ISI after a fixed test flash in a normal subject (Fig. 62.1a), a patient with CRD (Fig. 62.1b), a patient with Stargardt disease (Fig. 62.1c), and a patient with RP (Fig. 62.1d), respectively. Figure 62.2 shows the relative recovery of the rod response to the test flash from saturation (A/Amax), where A is the derived rod response amplitude of the test flash at a particular time point (t = ISI) after the test flash (A(t) = Amax − Aprobe(t)) (Pepperberg et al. 1997). The relative recovery of rod photoresponse was fitted with an exponential recovery function (A/Amax = exp[−(t − Tsat)/τ]), where Tsat indicates the initiation of rod recovery from saturation from the test flash. Technically, Tsat is the horizontal intercept of the exponential fit. In Fig. 62.2, the Tsat derived from the patient with RP is 254 ms, which is shorter than that derived from the normal subject (Tsat = 555 ms), the patient with CRD (Tsat = 523 ms) and the patient with Stargardt disease (Tsat = 580 ms).

Fig. 62.1.

Fig. 62.1

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Representative photoresponse recovery mediated by rod photoreceptors in normal subject and retinal degenerative diseases (RDDs). Representative rod-only waveforms generated by a probe flash at various inter-stimulus-interval (ISI) after a fixed test flash recorded from a normal subject (a), a patient with cone–rod dystrophy (CRD) (b), a patient with Stargardt disease (c), and a patient with RP (d). The rod-mediated photoresponse shows little recovery at ISIs of 500–600 ms in normal, CRD, and Stargardt disease (a–c). However, by an ISI of 500 ms, the rod-mediated photoresponse is fully recovered in the patient with RP

Fig. 62.2.

Fig. 62.2

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Deriving Tsat from patients with RP, CRD, and Stargardt disease. Tsat was derived from the rod-mediated photoresponse recovery shown in Fig. 62.1 using an exponential fit model (A/Amax = exp[−(t − Tsat)/τ]) (Birch et al. 1995; Pepperberg et al. 1997). Tsat derived from the patient with RP (Tsat = 254 ms) is shorter than normal (Tsat = 555 ms). However, Tsat derived from patients with CRD (Tsat = 523 ms) and Stargardt disease (Tsat = 580 ms) is comparable to normal (Tsat = 555 ms)

Figure 62.3 and Table 62.1 show Tsat values for 13 normal subjects, 5 patients with CRD, 4 patients with Stargardt disease, and 18 patients with adRP. Tsat derived from 5 patients with CRD was 473 ± 113 (SD) ms, which is not different from the normal 544 ± 92 (SD) ms (P = 0.26) (Fig. 62.3; Table 62.1). Tsat derived from 4 patients with Stargardt disease was 491 ± 98 (SD) ms, which is not different from the normal 544 ± 92 (SD) ms (P = 0.38) (Fig. 62.3; Table 62.1). However, Tsat derived from 18 patients with adRP was 331 ± 99 (SD) ms, which is 39% shorter from the normal average (P < 0.001) (Fig. 62.3; Table 62.1). Two patients with adRP showed Tsat values (489 and 520 ms) within the 99% confidence interval (CI) of normal (Fig. 62.3). These two highest Tsat values were derived from two patients who carried peripherin/rds mutation but retained normal ERG amplitudes at the time of pair-flash ERG recording.

Fig. 62.3.

Fig. 62.3

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Tsat derived from normal subject and RDDs. Tsat derived from 13 normal subjects, 5 patients with CRD, 4 patients with Stargardt disease, and 18 patients with autosomal dominant retinitis pigmentosa (adRP). Average value and standard deviation of each group is shown next to the scatter plot. Broken line: 99% confidence interval of normal Tsat distribution

Table 62.1.

Characterization of rod recovery kinetics with Tsat in patients with RDDs and normal subjects

Count Tsat (ms) Reduction Significance (P)
Normal 13 544 ± 92 (SD)
CRD   5 473 ± 113 0.26
Stargardt   4 491 ± 98 0.38
adRP 18 331 ± 99 39% <0.001
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This table presents the statistical results of Tsat in patients with CRD, Stargardt disease, or adRP. Statistical significance was calculated using two-tailed unpaired student t-test between sampled normal subjects and patients with RDDs

62.3.2 Tsat Is Correlated with Dark-Adapted Rod Amplitude in adRP

The ISCEV standard dark-adapted ERG rod response has been recognized as a surrogate marker for rod activity (Marmor et al. 2009). Figure 62.4 shows the relationship between Tsat and the ISCEV standard rod response amplitude in the 18 patients with adRP. A significant correlation exists between these two indices of rod dysfunction in patients with adRP (Pearson’s Correlation Coefficient = 0.54, P = 0.01) (Fig. 62.4). Thus, as ISCEV rod response diminishes, Tsat generally becomes shorter in patients with adRP. All patients with adRP with reduced rod amplitude also showed faster than normal recovery times.

Fig. 62.4.

Fig. 62.4

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Relationship between Tsat and the ISCEV standard rod response amplitude in patients with CRD, Stargardt disease, and adRP. A significant correlation (solid line) exists between these two indices of rod dysfunction in 18 adRP (Pearson’s Correlation Coefficient = 0.54, P = 0.01). These indices do not appear to be related in patients with CRD or Stargardt disease. Broken line: lower limit of normal Tsat and ISCEV rod amplitude (75 μV)

The results from the patients with CRD (solid triangles) and Stargardt disease (solid circles) suggest that the correlation does not hold in these diseases (Fig. 62.4). Note that three of these patients exhibited normal Tsat values despite rod amplitudes less than 75 μV, the lower limit of normal adult subjects (Birch and Anderson 1992).

62.4 Discussion

Previously, we reported that rods in retinitis pigmentosa, regardless of the inheritance pattern, recover earlier than normal (Wen et al. 2011). In this study, we found that patients with CRD and Stargardt disease show normal rod photoresponse recovery kinetics. This reinforces the suggestion that shortened rod photoresponse recovery is a result of primary rod degeneration in RP. This finding is consistent with previous invasive studies in laboratory animals. Kraft et al. (2005) showed that transgenic rods with pro-347-leu and pro-347-ser rhodopsin, both of which associated with human adRP, have earlier-than-normal time-to-peak, diminished sensitivity to light, and reduced integration time. In addition, it has been reported that rods with the Mertk mutation, which is associated with autosomal recessive RP in human (Gal et al. 2000), showed earlier-than-normal recovery (Niculescu 2004; Wen et al. 2006). Furthermore, rods that survived transient light damage in albino rat exhibited earlier-than-normal recovery (Wen 2008; Wen and Kraft 2008). Contrary to shortened rod recovery in RP, prolonged rod photoresponse recovery was observed in a type of cone dystrophy associated with GCAP1 mutations (Jiang et al. 2008). In this study, we found that CRD caused by ABCA4 mutations (Tsat = 523 and 499 ms) is not associated with either shortened or elongated rod photoresponse recovery.

Rod photoresponse recovery kinetics can be manipulated by genetic engineering in laboratory animals (He et al. 1998; Chen et al. 2000; Kraft et al. 2006; Krispel et al. 2006; Tsang et al. 2007). Moreover, it has been shown that rods are able to recover to normal recovery kinetics in a transient light damage model (Wen 2008; Wen and Kraft 2008). Because normal Tsat is specific to healthy rods, rod recovery kinetics could serve as biomarker for treatment efficacy in clinical studies and trials.

Acknowledgments

We thank Dr. Dianna Hughbanks-Wheaton and Kaylie Clark at the Southwest Eye Registry and the Daiger lab at UT Houston for coordinating and performing genetic testing. This investigation was supported by US National Institutes of Health grant (NEI R01 09076) to D.G.B and D.C.H. and the Foundation Fighting Blindness.

Contributor Information

Yuquan Wen, Email: ywen@retinafoundation.org, Rose-Silverthorne Retinal Degenerations Laboratory, Retina Foundation of the Southwest, 9900 N Central Expressway, Suite 400, Dallas, TX 75231, USA.

Kirsten G. Locke, Rose-Silverthorne Retinal Degenerations Laboratory, Retina Foundation of the Southwest, 9900 N Central Expressway, Suite 400, Dallas, TX 75231, USA

Donald C. Hood, Department of Psychology and Ophthalmology, Columbia University, New York, NY, USA

David G. Birch, Rose-Silverthorne Retinal Degenerations Laboratory, Retina Foundation of the Southwest, 9900 N Central Expressway, Suite 400, Dallas, TX 75231, USA Department of Ophthalmology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.

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