Vincent T. Ciavatta

Retinal expression of Fgf2 in RCS rats with subretinal microphotodiode array.

PURPOSE To test the hypothesis that subretinal electrical stimulation from a microphotodiode array (MPA) exerts a neuroprotective effect in Royal College of Surgeons (RCS) rats through the induction of growth factors. METHODS At postnatal day 21, RCS rats were divided into four groups in which one eye per rat received treatment: (A) active MPA, (M) minimally active MPA, (S) sham surgery, or (C) no surgery and the opposite eye was unoperated. Dark- and light-adapted ERGs were recorded 1 week after surgery. A second set of A-, M-, and C-treated RCS rats had weekly ERG recordings for 4 weeks. Real-time RT-PCR was used to measure relative expression of mRNAs (Bdnf, Fgf2, Fgf1, Cntf, Gdnf, and Igf1) in retina samples collected 2 days after the final ERG. RESULTS One week after surgery, there was a slight difference in dark-adapted ERG b-wave at the brightest flash intensity. Mean retinal Fgf2 expression in the treated eye relative to the opposite eye was greater for the A group (4.67 +/- 0.72) than for the M group (2.80 +/- 0.45; P = 0.0501), S group (2.03 +/- 0.45; P < 0.01), and C group (1.30 +/- 0.22; P < 0.001). No significant change was detected for Bdnf, Cntf, Fgf1, Gdnf, and Igf1. Four weeks after surgery, the A group had significantly larger dark- and light-adapted ERG b-waves than for the M and C groups (P < 0.01). Simultaneously, mean relative Fgf2 expression was again greater for the A group (3.28 +/- 0.61) than for the M (1.28 +/- 0.32; P < 0.05) and C (1.05 +/- 0.04; P < 0.05) groups. CONCLUSIONS The results show subretinal implantation of an MPA induces selective expression of Fgf2 above that expected from a retina-piercing injury. Preservation of ERG b-wave amplitude 4 weeks after implantation is accompanied by elevated Fgf2 expression. These results suggest that Fgf2 may play a role in the neuroprotection provided by subretinal electrical stimulation.

Natural and Somatic Embryo Development in Loblolly Pine: Gene Expression Studies Using Differential Display and DNA Arrays

In production biological systems, monitoring and controlling the growth environment is possible, but assessing the metabolic competency of the organism is more difficult. Somatic embryogenesis (SE), a tissue-culture method for multiplying embryos asexually, has great potential to capture at low cost the genetic gain from breeding and genetic-engineering programs. Loblolly pine, however, has proven recalcitrant in the production of somatic embryos suitable in quality for operational use. Many similarities and differences in gene expression were uncovered. We have modified a recent technique called differential display (DD) to examine gene expression, allowing the comparison of somatic and zygotic embryos. Over 400 cDNA “bands” have been cloned and their sequences determined. These bands can serve as “expression markers,” providing rapid, simple, and sensitive assessment of embryo physiology and development. These techniques are applicable to many areas of research where monitoring specific levels of gene expression is important for evaluating the performance of a system.

A promoter from the loblolly pine PtNIP1;1 gene directs expression in an early-embryogenesis and suspensor-specific fashion

Abstract. The PtNIP1;1 gene encodes an aquaglyceroporin that is expressed early in embryogenesis and appears to be expressed preferentially in the suspensor [V.T. Ciavatta et al. (2001) Plant Physiol 127:211–224]. An 899-bp fragment 5′ to the PtNIP1;1 open reading frame (NIP-899) was cloned from loblolly pine (Pinus taeda L.) genomic DNA and fused to the β-glucuronidase (GUS) reporter gene. The resulting plasmid, pNIP-GUS, was transformed into Norway spruce (Picea abies L.) embryogenic cultures by co-bombarding with a plasmid containing a bar gene construct as a selectable marker. The identity of lines selected on medium containing the herbicide Basta and showing β-glucuronidase activity was confirmed by polymerase chain reaction as harboring GUS. Histochemical GUS assays of these lines revealed GUS activity in all cells of proembryogenic masses. During early embryogeny, GUS staining was intense in the suspensor region but not detectable in embryonal masses. GUS staining was absent by mid-embryogeny. By contrast, a control transgenic line, transformed with EuCAD-GUS, expressed GUS throughout embryo development. These results suggest that NIP-899 contains elements that drive early embryogenesis-specific expression and suspensor-specific expression. This is the first example of a suspensor-specific promoter in conifers.

Natural and Somatic Embryo Development in Loblolly Pine

In production biological systems, monitoring and controlling the growth environment is possible, but assessing the metabolic competency of the organism is more difficult. Somatic embryogenesis (SE), a tissue-culture method for multiplying embryos asexually, has great potential to capture at low cost the genetic gain from breeding and genetic-engineering programs. Loblolly pine, however, has proven recalcitrant in the production of somatic embryos suitable in quality for operational use. Many similarities and differences in gene expression were uncovered. We have modified a recent technique called differential display (DD) to examine gene expression, allowing the comparison of somatic and zygotic embryos. Over 400 cDNA “bands” have been cloned and their sequences determined. These bands can serve as “expression markers,” providing rapid, simple, and sensitive assessment of embryo physiology and development. These techniques are applicable to many areas of research where monitoring specific levels of gene expression is important for evaluating the performance of a system.

Effects of Subretinal Electrical Stimulation in Mer-KO Mice

PURPOSE Subretinal electrical stimulation (SES) from microphotodiode arrays protects photoreceptors in the RCS rat model of retinitis pigmentosa. The authors examined whether mer(kd) mice, which share a Mertk mutation with RCS rats, showed similar neuroprotective effects from SES. METHODS Mer(kd) mice were implanted with a microphotodiode array at postnatal day (P) 14. Weekly electroretinograms (ERGs) followed by retinal histology at week 4 were compared with those of age-matched controls. RT-PCR for fibroblast growth factor beta (Fgf2), ciliary nerve trophic factor (Cntf), glial-derived neurotrophic factor (Gdnf), insulin growth factor 1 (Igf1), and glial fibrillary acidic protein (Gfap) was performed on retinas at 1 week after surgery. Rates of degeneration using ERG parameters were compared between mer(kd) mice and RCS rats from P28 to P42. RESULTS SES-treated mer(kd) mice showed no differences in ERG a- and b-wave amplitudes or photoreceptor numbers compared with controls. However, the expression of Fgf2 and Cntf was greater (6.5 ± 1.9- and 2.5 ± 0.5-fold, respectively; P < 0.02) in SES-treated mer(kd) retinas. Rates of degeneration were faster for dark-adapted maximal b-wave, log σ, and oscillatory potentials in mer(kd) mice than in RCS rats. CONCLUSIONS Although SES upregulated Fgf2 in mer(kd) retinas, as reported previously for RCS retinas, this was not accompanied by neuroprotection of photoreceptors. Comparisons of ERG responses from mer(kd) mice and RCS rats across different ages showed inner retinal dysfunction in mer(kd) mice but not in RCS rats. This inner retinal dysfunction and the faster rate of degeneration in mer(kd) mice may produce a retinal environment that is not responsive to neuroprotection from SES.

Neuroprotective Effects of Low Level Electrical Stimulation Therapy on Retinal Degeneration

Low-level electrical stimulation applied to the eye has been shown to have neuroprotective effects on photoreceptors and retinal ganglion cells. In this review, we compare the effects of Subretinal Electrical Stimulation (SES), Transcorneal Electrical Stimulation (TES), and Whole Eye Stimulation (WES) on preserving retinal structure and function, and visual acuity, in retinal degeneration. Similarities and differences in stimulus parameters, targeted cells and growth factor expression will be discussed with emphasis on studies that have translated laboratory findings into clinical trials.

Neuroprotective Dose Response in RCS Rats Implanted with Microphotodiode Arrays

Neuropreservation of retinal function and structure in RCS rats following implantation of a microphotodiode array (MPA) has been shown in previous studies (Pardue et al. J Neural Eng 2005;2:S39–47; Pardue et al. Invest Ophthalmol Vis Sci 2005;46:674–682). Since microphotodiodes produce electrical currents in proportion to the intensity of incident light, increased light exposure may result in greater neuroprotective effects. Our previous studies suggested that the frequency of light exposure to electroretinogram (ERG) flash stimuli might provide increased neuroprotection. Thus, in this study, we examined the dose response of subretinal electrical stimulation by exposing RCS rats implanted with MPAs to variable durations and combinations of two different lighting regimens: pulsing incandescent bulbs and xenon stimuli from an ERG Ganzfeld. While incandescent light regimens did not produce any significant differences in ERG function, we found significantly greater dark-adapted ERG b-wave amplitudes in RCS rats that received weekly vs. biweekly ERGs over the course of 8 weeks of follow-up. These results suggest that subretinal electrical stimulation may be optimized to produce greater neuroprotective effects by dosing with periodic higher current.