|Molecular Vision 2005;
Received 14 September 2004 | Accepted 28 April 2005 | Published 2 June 2005
Measurement of retinal injury in the rat after optic nerve transection: An RT-PCR study
Glyn Chidlow,1 Robert
J Casson,1 Paloma Sobrado-Calvo,2 Manuel Vidal-Sanz,2
Neville N. Osborne1
1Nuffield Laboratory of Ophthalmology, University of Oxford, Oxford, UK; 2Laboratorio de Oftalmologia Experimental, Universidad de Murcia, Murcia, Spain
Correspondence to: Dr. Glyn Chidlow, Nuffield Laboratory of Ophthalmology, University of Oxford, Walton Street, Oxford, OX2 6AW, UK; Phone: (01865) 248996; FAX: (01865) 794508; email: firstname.lastname@example.org
Purpose: In the current study, a non-histological approach, namely semi-quantitative RT-PCR, was used to provide information on retinal ganglion cell (RGC) injury and survival after optic nerve transection (ONT). The levels of mRNAs synthesized by RGCs and glial components were initially measured at defined time points after ONT. Subsequently, a comparison was made between the levels of these mRNAs in the ONT retinas of rats treated with the neuroprotectant BDNF and in rats which received vehicle.
Methods: Wistar rats received an ONT in one eye, while the fellow eye served as a control. ONT was performed 1-2 mm from the optic disc without damaging the retinal blood supply. In the first experiment, rats were killed at 1, 3, 5, 7, and 21 days after ONT. In the second experiment, brain derived neurotrophic factor (BDNF; 5 μg) or vehicle was injected intravitreally at the same time as the ONT and animals were killed after 7 days.
Results: After ONT, mRNA levels of RGC markers (NF-L and Thy-1) decreased substantially, while levels of GFAP and certain trophic factors mRNAs increased significantly. Administration of BDNF resulted in a substantial, but not complete, preservation of the levels of the RGC specific mRNAs, while ONT induced increases in GFAP and trophic factor mRNAs were not reduced to any great extent by BDNF.
Conclusions: The present studies show that measurement of NF-L and Thy-1 mRNAs provides a sensitive and reliable index of RGC injury after ONT, while measurement of GFAP and trophic factors mRNAs provides more general information on the effect of the injury on the retina.
Death of retinal ganglion cells (RGCs) is the cause of blindness in several diseases, including glaucoma. In recent years, much effort has gone into attempting to elucidate the causes and mechanisms of RGC loss so that strategies can be developed to counteract the process . Optic nerve transection (ONT) is a valuable model not only for investigation into pathways that contribute to RGC death but also as a model for neuronal apoptosis in the CNS . Death of RGCs does not commence until approximately 5 days following ONT, then, between day 5 and day 14, there is massive loss of RGCs and by day 14 only approximately 15% of the RGCs remain in the retina [3-5]. A variety of substances have been shown to attenuate RGC death after ONT, with brain derived neurotrophic factor (BDNF) receiving particular attention. Several studies have demonstrated that administration of BDNF delays RGC death after ONT, but importantly, even when the substance is administered chronically, the protection is only short lived [5-9].
Quantification of RGC injury following defined insults is a prerequisite in order to allow a comparison to be made between the effectiveness of potential neuroprotective drugs. In the CNS as a whole, neuroprotection is typically determined by quantifying the degree of cell loss. This is despite the fact that important reservations have been voiced concerning the limitations of a purely histological approach . The most common methodology utilized presently in the retina is retrograde labeling of RGCs with a tracer molecule such as fluorogold prior to performing the insult, with subsequent counting of surviving RGCs . However, there are certain drawbacks inherent in this approach: firstly, the procedure is only able to determine the effect of the potential neuroprotectant on the number of RGCs; it is unable to detect whether these remaining RGCs are dysfunctional or healthy, secondly, the effect of the insult and neuroprotectant on the surrounding cell types is unknown, and thirdly, only a small, predetermined area of the retina is usually studied and extrapolations of RGC numbers are made from counts that include only a small percentage of the total RGC population. In an attempt to provide an alternative and complementary approach, we developed a semiquantitative methodology to assess RGC injury in the whole of the retina after ischemia-reperfusion and excitotoxic injuries [11,12]. In this method, injury and subsequent death of RGCs can be followed by measurement of retinal mRNA levels of two markers specific to RGCs, namely Thy-1 and neurofilament light (NF-L). The method has proved reliable and sensitive [13,14] and similar strategies have been developed for analysis of brain tissue [15,16].
An important finding regarding the levels of RGC markers after certain injuries, which was first revealed by Hoffman et al.  and subsequently confirmed by Schlamp et al.  and by our group , is that downregulation of Thy-1 and NF-L mRNAs occurs in advance of detectable RGC death. Thus, in the period following the insult but prior to histological observation of cell loss, reduced levels of NF-L and Thy-1 can be viewed as an indicator of RGCs that are unhealthy and in all probability dysfunctional. Beside changes to RGCs, injuries such as ONT also cause activation of the retina's endogenous protection system. Precise knowledge of the various mechanisms that form this response is lacking, but upregulation of certain glial cell derived markers such as GFAP and trophic factors are one such event. It can therefore be argued that measurement of the levels of RGC markers, and of factors such as GFAP and FGF-2, in the period prior to and during cell loss should provide an accurate indication as to the health status of the retina in general and of RGCs in particular.
The purpose of this study was to assess the potential of RT-PCR as a tool for evaluating RGC injury and neuroprotection in the rat after ONT. Firstly, the levels of mRNAs synthesized by RGCs and glial components were measured at certain defined time points after ONT. Subsequently, a comparison was made between the levels of these mRNAs in the ONT retinas of rats treated with BDNF or vehicle. Since BDNF merely delays rather than prevents cell death, it is of considerable interest to discover what effect the compound has on the levels of these markers.
Treatment of animals
All experiments conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Adult Sprague-Dawley rats (200-250 g) were housed in a temperature and humidity controlled room with a 12 h light: 12 h dark cycle and provided with food and water ad libitum. For experimental manipulations, animals were anesthetized with an intraperitoneal injection mixture of Xylazine (Rompun; Bayer AG, Kiel, Germany) and Ketamine (Ketalar; Parke-Davis, Pontypool, UK), 10-15 and 30-100 mg/kg body weight, respectively. Two separate experiments were performed in this study: in the first experiment, various retinal mRNA levels were measured at prescribed time points after ONT, while in the second experiment, the effect of BDNF injection on ONT induced mRNA changes was investigated.
ONT was performed in one eye as described previously . In brief, to access the optic nerve, an incision was made in the skin covering the superior orbital rim, the superior orbital contents were dissected and the superior and external muscles were sectioned. Following rotation of the eye, the dura mater sheath was longitudinally opened and the optic nerve was completely transected as close as possible to the eye with care taken to avoid damage to the retinal blood supply. Rats were killed at various time points after ONT and the retinas removed for mRNA analysis. In a preliminary experiment, the fellow eye received a sham ONT. However, the sham operation was found to have no effect on any of the mRNAs analyzed (Figure 1) and in all subsequent experiments the fellow eye was untreated.
In order to investigate the effectiveness of BDNF after ONT, 16 rats were randomly assigned to one of two groups: one group of 8 rats received a single injection of BDNF (human BDNF; PeproTech, London, UK; 5 μg in 5 μl of PBS containing 1% bovine serum albumin) into the vitreous humor one h before the ONT, while the second group received vehicle solution. All rats were killed after 7 days.
For RT-PCR studies, retinas were carefully dissected, total RNA was isolated using Tri-reagent (Sigma, Poole, UK) according to the manufacturer's instructions and first strand cDNA synthesis was performed on 2 μg DNase treated RNA as described previously .
For conventional RT-PCR, the individual cDNA species were amplified in a reaction containing the cDNA equivalent of 25 ng total RNA, PCR buffer, MgCl2 (5 mM for NF-L, 4.5 mM for GAPDH and 4 mM for all other primers), dNTPs, the relevant sense and antisense primers and AmpliTaq Gold (Applied Biosystems, Warrington, UK). Reactions were initiated by incubating at 94 °C for 10 min, and PCRs (94 °C, 12 s; annealing temperature, 30 s; 72 °C, 30 s) performed for a suitable number of cycles (Table 1) followed by a final extension at 72 °C for 3 min. Interexperimental variations were avoided by performing all amplifications in a single run. All of the PCR products yielded single bands corresponding to the expected molecular weights to the expected size in base pairs. (Table 1) and PCR products were sequenced (data not shown) to ensure their validity. The oligonucleotide primer sequences and their annealing temperatures are shown in Table 1. PCR reaction products were separated on 1.5% agarose gels using ethidium bromide for visualization. The relative abundance of each PCR product was determined by quantitative analysis of digital photographs of gels using Labworks software (UVP Products, Upland, CA).
Prior to semi-quantitative amplification of the experimental samples, the amount of cDNA in all of the samples was equalized. In addition, the optimal conditions (e.g., Mg2+ concentration, annealing temperature) for each set of primers were determined. Subsequently, cycle-dependent reactions were performed for each mRNA species, in order to determine the linear range of detection by ethidium bromide. Once the linear range was established, PCRs were performed at the lowest cycle number that gave a reliably detectable product. To minimize variability, duplicate runs were performed for each mRNA amplified and the data were averaged.
For assessment of the levels of the various mRNAs in the retina, all values were normalized to that of the housekeeping gene GAPDH. GAPDH, thus, acted as an internal standard to correct for any variations in RNA isolation and/or cDNA synthesis. All results are expressed as mean±standard error of the mean (SEM) and differences between control and treated retinas were assessed using Student's paired t-test. Differences between treatment groups in the BDNF and IPC experiments were assessed using Student's unpaired t-test. In both cases, p<0.05 was considered statistically significant.
Real-time RT-PCR reactions were carried out in 96 well optical reaction plates using the cDNA equivalent of 50 ng total RNA for each sample in a total volume of 25 μl using the TaqMan Universal PCR Master Mix (Applied Biosystems, Warrington, UK). The thermal cycling conditions of PCR were as follows: 50 °C for 2 min, 95 °C for 10 min and 40 cycles of amplification comprising 95 °C for 15 s and 60 °C for 1 min. The PCR assay was performed using the ABI Prism 7700 Sequence detector (Applied Biosystems). NF-L forward and reverse primers (5'-CAC CGA CCG CCA CCA T-3' and 5'-CAC GTA GCG CCG CTT GT-3', respectively) and probe (5'-CGT TCA GCT ACG AGC CGT ACT TTT CGA C-3') were selected from Genbank and designed using the primer design software Primer Express (Applied Biosystems), while GAPDH primers and probe were obtained from TaqMan rodent GAPDH control reagents (Applied Biosystems). Amplification of GAPDH mRNA was performed as the internal control gene to account for variations in RNA levels between different samples. In order to allow a comparison to be made between the levels of expression of NF-L in vehicle and BDNF treated retinas, results obtained from the real-time PCR experiments were quantified using the comparative threshold (2-ΔΔCT) method . This method essentially comprises the same two steps as for conventional RT-PCR: firstly, the amount of NF-L is normalized to the levels of the endogenous housekeeping gene (GAPDH), and secondly, the normalized amount of NF-L in the axotomized retina is then expressed relative to the normalized amount of NF-L in the control retina. The threshold cycles (CT) were calculated using ABI Prism 7000 SDS Software (Applied Biosystems).
Histopathology and immunohistochemistry
Following enucleation, whole eyes were fixed in 10% neutral buffered formalin and processed for routine paraffin embedded sections on an automated tissue processor. Eyes were embedded sagitally and 5 μm serial sections were cut using a rotary microtome. For histopathology, sections were stained with hematoxylin and eosin. For immunohistochemistry, tissue sections were deparaffinized, rehydrated into 70% ethanol and treated for 10 min with 0.5% H2O2 in absolute methanol to block endogenous peroxidases before being taken to deionized water. Antigen retrieval was achieved by incubating the slides in boiling 10 mM citrate buffer (pH 6.0) at a pressure of 5 psi for 5 min. After being washed in PBS-T (PBS containing 0.1% Triton X-100) tissue sections were then equilibrated for in PBS-T containing primary antibody. Sections analyzed for immunohistochemical localization of NF-L were incubated overnight at 4 °C with mouse anti-neurofilament light antibody (1:225; Chemicon, Chandlers Ford, UK). Following incubation with primary antiserum, sections were washed in PBS-T and incubated in a solution of PBS-T containing anti-mouse secondary antibody linked to biotin (Vector Labs., Peterborough, UK; 1:100) and horse serum (1:100) for 30 min. After a further wash with PBS-T, sections were developed using a standard avidin-peroxidase detection system (Vector Laboratories, Peterborough, UK) with 0.1% (v/v) H2O2 and 0.1% (w/v) 3'-,3'-diaminobenzidine in PBS-T. Sections were mounted in PBS-glycerol and visualized by light microscopy.
Histopathology and immunohistochemistry
Typical light photomicrographs of hematoxylin and eosin stained retinas taken 7 days after ONT are shown in Figure 1. In vehicle treated retinas, there appeared to be a selective loss of cells in the GCL (Figure 1B) compared to the control retina (Figure 1A). In BDNF treated retinas, this loss of cells was not apparent (Figure 1C). Due to the presence of an unknown number of displaced amacrine cells in the RGC layer and the difficulty in obtaining sections from exactly the same eccentricity, precise quantification of the numbers of cells in the GCL was not undertaken.
In order to confirm that NF-L is a marker for RGCs in the rat, NF-L immunohistochemistry was performed on transverse retinal sections. The results showed that NF-L labeling is associated with the somata and the axons of RGCs (Figure 1D,E) and with axons in the optic nerve head (Figure 1F), but is virtually absent from the other layers of the retina. The results correlated well with previous localization of NF-L immunoreactivity in the hamster , human and pig  retinas.
Changes in retinal mRNA levels after ONT
ONT followed by 21 days recovery caused substantial decreases in the total retinal levels of NF-L and Thy-1 mRNAs, but no change in the level of rhodopsin mRNA, relative to the control untreated retina (Figure 2). In order to verify that these differences are specific to axonal cut, and are not partially a response to the surgical procedure itself, a comparison was made between the mRNA levels and the a- and b-waves of the electroretinogram in ONT eyes, in eyes that received a sham operation and in untreated control eyes. It can be seen that the relative decreases in Thy-1 and NF-L mRNAs in ONT eyes were effectively identical, irrespective as to whether the contralateral eye was untreated or received a sham operation (Figure 2). Moreover, the sham procedure caused no changes in the levels of ganglion cell specific mRNAs and rhodopsin mRNA (Figure 2) or of GFAP and trophic factor mRNAs (data not shown) relative to control untreated eyes. The sham procedure was also found to have no effect on the electroretinogram (data not shown). As a consequence of these results, the contralateral eye was untreated in all subsequent ONT experiments. Analysis of the time courses of the decreases of NF-L and Thy-1 mRNAs revealed an excellent correlation between the levels of the two mRNAs at each time point (Figure 3). It can be seen that levels of both transcripts had begun to decline within 24 h of ONT, were substantially lower by 3 days, and that the decrease was close to maximal by 7 days, although some additional loss did occur during the remainder of the three week period.
The effect of ONT on the retinal levels of GFAP, FGF-2, CNTF, BDNF, NGF, and GDNF mRNAs is shown in Table 2. It can be seen that ONT caused a significant upregulation of all of the factors analyzed with the exception of GDNF. However, the time courses and magnitudes of the individual responses varied considerably. Upregulation of FGF-2 mRNA was first detectable after 3 days and continued to rise throughout the three week period analyzed, while expression of GFAP and CNTF increased during the first 7 days but then declined somewhat. The BDNF mRNA level was only elevated for the first 3 days following ONT, while the level of NGF mRNA was unchanged until 7 days, but remained higher than the control retina for the following 2 weeks.
Effect of BDNF on ONT induced mRNA changes
In vehicle injected rats, ONT followed by 7 days recovery caused an 84% reduction in the total retinal level of NF-L mRNA when measured by real-time RT-PCR (Figure 4A) and a 66% reduction when measured by conventional RT-PCR (Figure 4B). In contrast, in BDNF injected rats, the total retinal level of NF-L was only reduced by 37% when measured by real-time RT-PCR (Figure 4A) and by 26% when measured by conventional RT-PCR (Figure 4B). Thus, BDNF elicited 56.2% or 61.6% protection against ONT induced loss of NF-L mRNA when measured by real-time and conventional RT-PCR, respectively. These values were not statistically different from each other.
The magnitude of the ONT induced decrease in the Thy-1 mRNA level in retinas of vehicle treated rats was, as expected, almost indistinguishable from the decrease in NF-L mRNA (Figure 5A). At 7 days after ONT, BDNF likewise afforded significant protection against the loss of Thy-1 mRNA, although the extent of protection against Thy-1 loss was marginally less pronounced than for NF-L (Figure 5A). In vehicle treated rats, significant increases in the retinal levels of FGF-2, CNTF, NGF and GFAP mRNAs were all measured 7 days after ONT (Figure 5B,C). Even though the increases in all four of these mRNAs were less marked in the retinas of BDNF treated rats (Figure 5B,C), a statistical difference was only apparent for NGF mRNA.
Optic nerve transection (ONT) is a well established model for investigation into the pathways that contribute to RGC death, which has been utilized extensively to screen the effectiveness of potential neuroprotectants . To date, the principal method of determining reliably whether particular treatments aid RGC survival has been by counting cells prelabeled with a tracer molecule . In the current study, we investigated the possibility of using a non-histological approach, namely semi-quantitative RT-PCR, to provide information on RGC injury and survival after ONT. The results showed that measurement of NF-L and Thy-1 mRNAs provides an alternative index of RGC injury, which is both sensitive and reliable, while measurement of GFAP and trophic factors mRNAs provides more general information on the effect of the injury on the retina as a whole.
Measurement of RGC specific mRNAs after ONT
Before drawing conclusions about the effect of ONT on mRNA levels in the retina, it was important to establish that changes are specific to axonal cut and are not partially a response to any associated ischemia. This was achieved by demonstrating that there were no differences in the electroretinogram or in mRNA levels in sham operated and untreated eyes. Subsequently, we measured changes in NF-L and Thy-1 mRNA levels at various time points after ONT and correlated the results with the histological progression of RGC death. The time course of RGC loss after ONT has been well documented: cell death is first observed at 4-5 days, reaches 50% of the original population by day 7 and 85% by day 14 [3,4]. In contrast, NF-L and Thy-1 mRNA levels, which responded in an almost identical fashion, had begun to decline within 24 h of ONT and were more than 50% lower than the control retina after 3 days. The results are in agreement with those of Schlamp et al.  who noted that loss of Thy-1 mRNA precedes cellular death after optic nerve crush in mice. This indicates that the RGC soma is adversely affected by optic nerve injury for a considerable period before actual cell death and clearance of cell debris occurs. Downregulation of these mRNAs can therefore be viewed as an early functional marker of RGC injury.
The results we obtained are also in agreement with the in situ hybridization studies of McKerracher et al. [23,24]. In their work, McKerracher and colleagues showed that, within the GCL, there was a decrease in the intensity of the hybridization signal for neurofilament medium (NF-M) mRNA during the first week after axotomy, which remained low thereafter. To determine whether the reduced NF-M mRNA level detected after ONT represented a specific change in the expression of this cytoskeletal protein or a more general cellular response to the injury, the mRNA level of the housekeeping gene GAPDH within the GCL was also measured. The results showed that the intensity of the GAPDH signal had also markedly declined by 2 weeks after injury, and thus that axotomization causes extensive and persistent changes in both structural and energy-generating pathways within RGCs that ultimately lead to their death.
Previous studies have shown that a single intravitreal injection of BDNF at the time of ONT results in the survival of all RGCs for one week, but thereafter cell death occurs at the same rate as in vehicle treated animals [5,7]. Given that RGC soma downregulate NF-L and Thy-1 genes within three days of ONT, it was of interest to ascertain whether administration of BDNF maintains the levels of these mRNAs for 7 days. The data showed that BNDF did significantly preserve the levels of the RGC specific mRNAs after ONT, but that the degree of protection was not as pronounced as that obtained by counting cells [5,7]. The explanation for the difference is given by the recognition that measurement of RGC markers is a more sensitive index of cellular damage and that while BDNF does improve the health status of the cells in the short term, it does not prevent the chain of events occurring that eventually leads to cell death. Schlamp et al.  and our own group  have previously argued that measurement of RGC markers may represent a functional assay of the health status of RGCs after injury, which would be particularly useful when testing the efficacy of neuroprotective drugs. This argument appears to be validated by the current results.
The development of real-time RT-PCR has allowed a more sensitive and precise quantification of the relative levels of particular transcripts between samples. This is because real-time PCR determines the level of mRNA during the exponential phase of amplification whereas conventional PCR detects the mRNA level in the succeeding linear phase. We measured the level of NF-L mRNA in vehicle and BDNF treated retinal samples using conventional and real-time RT-PCR to see whether the latter technique offered greater accuracy or precision in detecting differences between the treatments. The results showed in fact that a good correlation exists between the two methodologies; the protection conferred by BDNF differed by only 5% when measured by each procedure. The finding validates the use of conventional RT-PCR for use in studies such as these. Nevertheless, it should be noted that although the degree of protection was similar using both methods, the actual decrease in NF-L mRNA after ONT was slightly greater when measured by real-time PCR. This is explained by recognition of the fact that detection of PCR product yields by the agarose gel/ethidium bromide/densitometry method typically employed for conventional PCR offers lower resolution and sensitivity than the detection system incorporated into the real-time PCR system. Accordingly, conventional RT-PCR overestimated somewhat the amount of mRNA in samples with a low amount of NF-L. With regard to variability, the real-time data displayed only slightly (approximately 2%) less variability between samples than the data obtained via conventional PCR, which further emphasizes the legitimacy of both techniques. Employment of a more modern dye than ethidium bromide, such as SYBR Green, may offer increased sensitivity and a greater chance of detecting small differences in mRNA expression between samples.
Internal controls are critical in semiquantitative PCR to normalize for variations in mRNA levels between different samples. A number of different internal endogenous controls are used in studies of gene expression, including GAPDH, cyclophilin, β-actin, tubulin and ribosomal RNA. As discussed by a number of authors [25,26], the prime requirement for any control transcript is maintenance of a consistent level of expression under the experimental conditions. GAPDH is probably the most widely used control transcript, but its expression, like that of β-actin, can vary under a variety of circumstances. A number of authors advocate the use of 18S or 28S rRNA as internal standards; however, since these are ribosomal RNAs, they are not always representative of the mRNA population. To summarize, no one control is ideal and selection will depend on the system being studied. GAPDH was chosen as the internal control in this study because its level of expression is unaltered after ischemia/reperfusion in the rat  and appears unchanged following ONT . In much of the study, we also used cyclophilin and rhodopsin as internal controls (data not shown) and found that they gave similar results to GAPDH. Rhodopsin mRNA, which is expressed constitutively at a high level in the retina, is known to be unaffected in the early period following ONT and can function as a useful endogenous control transcript under a variety of circumstances where photoreceptors are not affected.
Measurement of Trophic factor mRNAs after ONT
One theory put forward to account for the death of RGCs after ONT is that axotomy results in the loss of target derived neurotrophic factors . Much evidence has been accumulated in support of this hypothesis, for example, data showing that exogenous administration of various trophic factors delays RGC loss after ONT. Interestingly, endogenous expression of certain trophic factors increases after ONT [28-30]. These increases are suggested to form part of the innate response of the retina to injuries such as ONT, and come about largely through activation of Müller cells. Knowledge of the temporal sequence in which the different neurotrophic factors are upregulated may shed light on the survival pathways that are activated following ONT and why, ultimately, they fail to prevent RGC death. Moreover, we hypothesized that measuring the levels of GFAP and neurotrophic factor mRNAs after ONT would provide an indication as to the severity of neuronal injury, and that reduced levels of these endogenous survival factors would be present in retinas that had received neuroprotectant therapy.
In agreement with the results of previous studies, which have demonstrated increased protein levels of CNTF [29,30], FGF-2 , and GFAP  in the retina one week after ONT, we showed that the mRNAs encoding GFAP, FGF-2, and CNTF are all likewise elevated at this time point. In addition, we showed that the transcript for NGF, but not for BDNF or GDNF, is also significantly upregulated 7 days after ONT, although the magnitude of this response was less than for the others. Curiously, the earliest, most efficacious, and longest lasting response was by FGF-2, which is the least effective of these trophic factors when administered exogenously. It is conceivable that the robustness of the endogenous FGF-2 response negates the effectiveness of exogenously applied FGF-2. In retinas treated with the neuroprotectant BDNF, the ONT induced increases in FGF-2, CNTF, GFAP, and NGF mRNAs were all somewhat diminished compared with vehicle treated retinas, although only NGF significantly so. The result indicates that the Müller cells respond to the effects of the applied BDNF and modify their production of survival factors correspondingly. However, the relatively modest reductions in the levels of CNTF, FGF-2, and GFAP after BDNF treatment confirms that the Müller cells are also sensitive to the fact that BDNF will only afford temporary rescue to RGCs from the ongoing injury process.
Gao et al.  showed that optic nerve crush increased BDNF mRNA expression, which peaked at 48 h then declined to the basal level. In situ hybridization revealed it is the RGCs themselves, and not Müller cells, that upregulate the BDNF synthesis, and the authors suggested that the effect is an endogenous survival mechanism of the injured retina. In the current study, we showed that a similar response occurs after ONT. The BDNF mRNA level in the retina was elevated in the first 72 h after ONT, however, the effect had been lost by 7 days. It can be concluded that while elevated BDNF may be partly responsible for delaying the death of RGCs by a number of days, it is clearly insufficient to prevent the subsequent catastrophic loss of cells. The results correlate well with data showing that exogenous BDNF only delays loss of cells by a short period of time [5,7,8].
In contrast to the other trophic factors, the results obtained for GDNF were unexpected. A number of recent studies have demonstrated that GDNF promotes the survival of axotomized RGCs [33-35]. What is less clear is whether GDNF forms part of the endogenous response of the retina to optic nerve injury: Koeberle and Ball  showed that GDNF immunoreactivity is localized to RGCs and photoreceptors in the normal retina and is decreased following ONT. However, Lindqvist et al.  detected an upregulation of GDNF mRNA in the period after ONT. In the current study, in which we likewise measured GDNF mRNA after ONT, we did not observe any change in the amount of transcript after ONT. The reason for the discrepancy between the two studies is unresolved but could conceivably be related to the different techniques utilized to measure mRNA, or to the fact that different strains of rat were used.
The work was supported by grant PRO AGE RET (QLK6-2000-00569) from the European Community. The authors are grateful to Dr. Mark Graham of AstraZeneca for the use of the ABI Prism 7700 Sequence detector. The authors are grateful to Maria E. Aguilera and Antonio García for technical assistance. This manuscript is dedicated to the late Dr. Jose Melena who also provided expert technical assistance during the study.
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