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Systemic administration of acidic fibroblast growth factor ameliorates the ischemic injury of the retina in rats
Neuroscience Letters 255 (1998) 1–4
Systemic administration of acidic fibroblast growth factor ameliorates the ischemic injury of the retina in rats Pedro Cuevas a ,*, Fernando Carceller a, Mariano Redondo-Horcajob, Rosa Maria Lozano b, Guillermo Gime´nez-Gallego b a
Departamento de Investigacio´n, Servicio de Histologı´a, Hospital Ramo´n y Cajal, E-28034 Madrid, Spain b Departamento de Quı´mica de Proteı´nas, CSIC, E-28006 Madrid, Spain Received 15 July 1998; received in revised form 6 August 1998; accepted 12 August 1998
Abstract The central neuroprotective effects against ischemic injury of fibroblast growth factor (FGF), administered either directly into the central nervous system or systemically, is well documented. Here we show in a rat model of transient retinal ischemia that the neuroprotective effect of systemically administered acidic fibroblast growth factor (aFGF, FGF-1) extends to the retina. Histological findings show a lower decrease of retinal ganglion cells and inner nuclear layer cells (P , 0.0001) in animals receiving FGF-1. These results suggest that FGF may function as a natural protection agent during transient retinal ischemia and further document that an efficient neuroprotection of central nervous tissues can be obtained by systemic administration of this protein. Our data may, thus, contribute to the development of novel and safe therapeutic approach for the treatment of the ischemic injury of the retina. 1998 Elsevier Science Ireland Ltd. All rights reserved
Keywords: Retinal ischemia; Rat; Fibroblast growth factor; Neuroprotection
Fibroblast growth factors (FGFs) have been shown to boost brain recovery after ischemia. The rationale for this effect was suggested by the fact that the levels of these proteins significantly increase following ischemic injury [19], which has been interpreted to reflect a physiological protection mechanism, since FGFs are powerful survival promoters for several neuronal populations [2,10,17,21]. The inability of these proteins to cross the blood–brain barrier (BBB) has been assumed to be due to their relatively high molecular mass. Consequently, it has been proposed that their administration to the central nervous system (CNS) requires invasive procedures which, obviously, constitute a serious limitation for a widespread clinical application. However, recently reported data suggest that FGFs cross the BBB [3] at an adequate level for protecting brain tissues from ischemic-reperfusion injury [4,5,11]. Recently, the enhancement of the expression of FGF and its receptor mRNAs has been reported in ischemic-reper* Corresponding author. Tel./fax: +34 91 3368290; e-mail:[email protected]
fused rat retina [16], data that have been interpreted as a physiological self-protective mechanism for minimizing tissue damage. Indeed, the intracameral administration of FGF preserves inner retinal layers from ischemia [20,23]. As we have shown that systemically injected FGF readily accumulates at the retina [3], a series of studies, reported here, aimed at establishing whether intravenously administered acidic fibroblast growth factor (FGF-1) might ameliorate the severity of retinal injury caused by transient ischemic episodes, were undertaken. The experiments were performed in accordance to the guidelines summarized in [24]. Retinal ischemia and reperfusion were induced according to the technique described by Stefansson et al. [18]. In brief, Sprague–Dawley rats (250–300 g) of either sex were anesthetized with an intraperitoneal injection of 3ml/kg of a mixture of ketamine hydrochloride (2.5 mg/ml), valium (2 mg/ml) and atropine (0.1 mg/ml). After a lateral conjunctival peritomy and desinsertion of the lateral rectus muscle, the optic nerve of the right eye was exposed. A 6–0 nylon suture was passed behind the optic nerve and was tightened until blood flow ceased in all retinal vessels.
0304-3940/98/$19.00 1998 Elsevier Science Ireland Ltd. All rights reserved PII S0304- 3940(98) 00672- 7
2
P. Cuevas et al. / Neuroscience Letters 255 (1998) 1–4
The complete absence of blood flow was assessed with an operating microscope with a flat contact lens. Ninety minutes after the ischemia onset, the suture was removed, and reperfusion of the retinal vessels monitored through the operating microscope. Eyes that failed to reperfuse within 5 min were excluded from this experiment. Recombinant human FGF-1 was synthesized according to method of Zazo et al. [22] and dissolved in PBS containing 0.1% heparin. A volume of 2.6 mg aFGF was injected in 50 ml solution through a jugular vein at the onset of reperfusion (n = 10). The dosage and time of FGF administration were selected on the basis of our previous demonstration that such a dose protects the rat brain during postischemic reperfusion [4]. Control animals (n = 10) received 50 ml of vehicle solution in the same manner. Sham-operated animals (n = 5) were manipulated in a similar fashion without retinal vessels occlusion. Two days after the ischemic episode the animals were reanesthetized and retrogradely perfused through a heparinized cannula introduced into the abdominal aorta via the femoral artery. For washing out the blood through the excised inferior vena cava, 200 ml PBS containing 0.1% heparin was used. Fixation was performed with 200 ml of 4% formaldehyde. Then the ischemic and nonischemic eyes were enucleated and divided into two halves by coronal section through the ora serrata. The vitreous was removed, and the posterior half of the eye was immersed in the same fixative for 24 h. Specimens were dehydrated and embedded in paraffin. Horizontal sections (4 mm thickness) were stained with hematoxylin and eosin. Ischemic damage was quantified in two ways. First, the thickness of the outer nuclear and outer plexiform layers (ONL and OPL) were measured in three different sections at 1 mm from both sides of the optic nerve and compared with values to that in the contralateral nonischemic eye. Second, in the same sections the number of cells in ganglion
cell layer (GCL) and inner nuclear layer (INL) were counted from one ora serrata to the other ora serrata and compared with GCL and INL cell counts in contralateral nonischemic eye. The changes in retinal layer thickness and the number of cells were expressed in percent ratios with respect to those of the nonischemic eye. The statistical significance was subsequently evaluated by ANOVA and Student’s ttests. Histopathological changes after transient eye ischemia were limited mostly to the inner part of the retina; only small alterations were seen in the photoreceptor layer using light microscopy. Fig. 1 shows the changes in retinal layers of the ischemic vehicle eye compared to FGF-1 ischemic eye. Quantification of those differences appears summarized in Fig. 2. According to these data, the inner plexiform layer (IPL) thickness of the ischemic eyes, which received vehicle solution, and those treated with FGF-1 were (mean ± SEM) 52.4 ± 5,75 and 82.7 ± 5.04% of the nonischemic ones, respectively. These differences are significant at the level of P , 0.0003 (Fig. 2A). There were no significant differences in eye layer thickness for the ONL, OPL and IPL between the ischemic and non ischemic eyes. As shown in Fig. 2B, the percentage of surviving neurons in GCL is significantly lower in vehicle-treated ischemic eyes than in FGF-1-treated ones (26.5 ± 2.89 vs. 61.0 ± 3.29%, P , 0.0001). Finally, the ratios of nuclei cell counts in INL between the ischemic and nonischemic eyes was higher in the FGF-1-treated group than in that which received vehicle (80.30 ± 3.67 vs. 45.3 ± 2.94%, P , 0.0001) (Fig. 2C). No morphological disturbances have been observed in sham-operated retina. Severe edema is observed in vehicle-treated animals (Fig. 1). This finding could explain that INL thickness is similar in vehicle- and FGF-1-treated animals although in the INL from ischemic vehicle retina the number of cells is lower than in INL from FGF-1-treated retina. In addition, vehicle-
Fig. 1. Light micrographs of the posterior ischemic rat retinas. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; ONL, outer nuclear layer; R and C, layer of rods and cones. An eye treated with vehicle solution at the onset of reperfusion shows an inner retina that is reduced in thickness compared to an FGF-1-treated eye. Note that the vehicle-treated retina shows an intense edema. Hematoxylin and eosin staining (original magnification ×50).
P. Cuevas et al. / Neuroscience Letters 255 (1998) 1–4
3
Fig. 2. Statistic assessment of the effect of systemic administration of F-1 on the histological structure of the retina after a transient ischemia episode. (A) Mean thickness of ischemic retinal IPL as a percentage of the mean thickness of retinal IPL of the nonischemic contralateral eye. The mean thickness of IPL was significantly greater in the FGF-1 than in the vehicle-treated eyes (P , 0.0003). (B) Ischemic retinal ganglion cell number as a percentage of retinal ganglion cells of the nonischemic contralateral eye. Retinal ganglion cell counts were significantly higher in the FGF-1-treated eyes than in vehicle-treated ones (P , 0.0001). (C) Cell counts in ischemic INL as a percentage of cell counts in nonischemic contralateral INL. The difference between the vehicle group and the group receiving FGF-1 was significant (P , 0.0001).
treated eyes show inflammatory signs in the inner retinal layers. The absence of inflammatory responses following transient retinal ischemia observed in FGF-1-treated eyes could be explained by the postulated anti-inflammatory activities of FGF [13]. This report shows that transient retinal ischemia in the rat eye causes a decrease in the IPL thickness and supports that this decrease is a reliable parameter for ischemic loss of retinal ganglion cells [8]. The results of the present study confirm and extend observations that have indicated that intracameral administration of FGF preserves the inner retinal layers from ischemia [20,23]. Although the nature of the protective effect of FGF against retinal injury and its relation to protection from cell death remains to be investigated, it has been shown that FGF protects against neurotoxicity during brain ischemia by a buffering Ca2 + mechanism [6,7,15]. One possible mechanism whereby FGF protects retina from reperfusion injury may be mediated by the capacity of FGF to suppress the expression of NMDA receptor protein [14]. Since retinal ischemia involves NMDA receptor activation [1], suppression of NMDA receptors can stabilize Ca2+ and thereby protect neurons against degeneration. It has been shown that intraocular FGF administration protects retinal tissue against ischemia [20,23]. However, this method of FGF delivery is not without potential side effects [12]. In this report we show retinal neuroprotection after transient ischemia using a single systemic injection of FGF. In addition, this finding adds further arguments in favor of FGF penetration across BBB and is in accordance with other studies showing neuroprotective effects of peripherally delivered FGF [9,11]. We thank Ch. Bourdier for editorial assistance and A. Ferna´ndez for technical aid. This work was supported by Fundacio´n Futuro. [1] Bresnick, G.H., Excitotoxins: a possible new mechanism for the
[2]
[3]
[4]
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pathogenesis of ischemic retinal damage, Arch. Ophthalmol., 107 (1989) 339–341. Cheng, B. and Mattson, M.P., NGF and bFGF protect rat hippocampal and human cortical neurons against hypoglucemic damage by stabilising calcium homeostasis, Neuron, 7 (1991) 1031–1041. Cuevas, P., Fernandez Ayerdi, A., Carceller, F., Collin, S., Mascarelli, F., Mun˜oz Willery, Y. and Gime´nez-Gallego, G., Central nervous system distribution of fibroblast growth factor injected into the blood stream, Neurol. Res., 18 (1996) 267– 272. Cuevas, P., Carceller, F. and Gime´nez-Gallego, G., Intravenous fibroblast growth factor penetrates the blood–brain barrier and protects hippocampal neurons against ischemia-reperfusion injury, Surg. Neurol., 49 (1998) 77–84. Cuevas, P., Carceller, F., Reimers, D., Saenz de Tejada, Y. and Gime´nez-Gallego, G., Acidic fibroblast growth factor rescues gerbil hippocampal neurons from ischemic apoptotic death, Neurol. Res., 20 (1998) 271–274. Freese, A., Finklestein, S.P. and DiFiglia, M., Basic fibroblast growth factor protects striatal neurons in vitro from NMDAreceptor mediated excitotoxicity, Brain Res., 575 (1992) 351– 355. Heidinger, V., Hicks, D., Sahel, J. and Dreyfus, H., Peptide growth factors but not ganglioside protect against excitotoxicity in rat retinal neurons in vitro, Brain Res., 767 (1997) 279– 288. Hughes, W.F., Quantitation of ischemic damage in the rat retina, Exp. Eye Res., 53 (1991) 573–582. Ikeda, K., Iwasaki, Y., Tagaya, N., Shiojima, T., Kobayashi, T. and Kinoshita, M., Neuroprotective effect of basic fibroblast growth factor on wobbler mouse motor neuron disease, Neurol. Res., 17 (1995) 445–448. Ishihara, A., Saito, H. and Nishiyama, N., Basic fibroblast growth factor ameliorates learning deficits in basal forebrainlesioned mice, Jpn. J. Pharmacol., 59 (1992) 7–13. Kirschner, P.B., Henshaw, R., Weise, J., Trubetskoy, V., Finklestein, S., Schultz, J.B. and Beal, M.F., Basic fibroblast growth factor protects against excitotoxicity and chemical hypoxia in both neonatal and adult rats, J. Cereb. Blood Flow Metab., 15 (1995) 619–623. Lavail, N.M., Unoki, K., Yasumura, D., Matthes, M.T., Yancopoulos, G.D. and Steinberg, R.H., Multiple growth factors, cytokines and neurotrophins rescue photoreceptors from
4
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P. Cuevas et al. / Neuroscience Letters 255 (1998) 1–4 the damaging effects of constant light, Proc. Natl. Acad. Sci. USA, 89 (1992) 11249–11253. Lefaucher, J.P., Gjata, B., Lafont, H. and Sebile, A., Angiogenic and inflammatory responses following skeletal muscle injury are altered by immune neutralization of endogenous basic fibroblast growth factor insulin-like growth factor-1 and transforming growth factor-b1, J. Neuroimmunol., 70 (1996) 37–44. Matson, M.P., Kumar, K., Cheng, B., Wang, H. and Michelis, E.K., Basic FGF regulates the expression of a functional 71 Kda NMDA receptor protein that mediates calcium influx and neurotoxicity in cultured hippocampal neurons, J. Neurosci., 13 (1993) 4575–4588. Mattson, M.P., Cheng, B. and Smith-Swintosky, V.L., Growth factor-mediated protection from excitotoxicity and disturbances in calcium and free radical metabolism, Semin. Neurosci., 5 (1993) 295–307. Miyashiro, M., Ogata, N., Takahashi, Kl., Matsushima, M., Yamamoto, C., Yamada, H. and Uyama, M., Expression of basic fibroblast growth factor and its receptor mRNA in retinal tissue following ischemic injury in the rat, Graefe’s Arch. Clin. Exp. Ophthalmol., 236 (1998) 295–300. Otto, D. and Unsicker, K., Basic FGF reverses chemical and morphological deficits in the nigrostriatal system of MPTP-treated mice, J. Neurosci., 10 (1990) 1912–1921. Stefansson, E., Wilson, C.A., Schoen, T. and Kuwabara, T.,
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Experimental ischemia induces cell mitosis in the adult rat retina, Invest. Ophthalmol. Vis. Sci., 29 (1988) 1050–1055. Takami, K., Iwane, M., Kiyota, Y., Miyamoto, M., Tsukuda, R. and Shiosaka, S., Increase of basic fibroblast growth factor immunoreactivity and its mRNA level in rat brain following transient forebrain ischemia, Exp. Brain Res., 90 (1992) 1–10. Unoki, K. and Lavail, M.M., Protection of the rat retina from ischemic injury by brain-derived neurotrophic factor, ciliary neurotrophic factor, and basic fibroblast growth factor, Invest. Ophthalmol. Vis. Sci., 35 (1994) 907–915. Walicke, P.A., Cowan, W.M., Ueno, N., Baird, A. and Guillemin, R., Fibroblast growth factor promotes survival of dissociated hippocampal neurons and enhances neurite extension, Proc. Natl. Acad. Sci. USA, 83 (1986) 3012–3016. Zazo, M., Lozano, R.M., Ortega, S., Varela, J., Diaz-Orejas, R., Ramirez, J.M. and Gime´nez-Gallego, G., High-level synthesis in Escherichia coli of shortened and full-length human acidic fibroblast growth factor and purification in a form stable in aqueous solutions, Gene, 113 (1992) 231–238. Zhang, C., Takahashi, K., Lam, T.T. and Tso, M.O.M., Effects of basic fibroblast growth factor in retinal ischemia, Invest. Ophathlmol. Vis. Sci., 35 (1994) 3163–3168. Zimmermann, M., Ethical principles for the maintenance and use of animals in neuroscience research, Neurosci. Lett., 73 (1987) 1.
Systemic administration of acidic fibroblast growth factor ameliorates the ischemic injury of the retina in rats Pedro Cuevas a ,*, Fernando Carceller a, Mariano Redondo-Horcajob, Rosa Maria Lozano b, Guillermo Gime´nez-Gallego b a
Departamento de Investigacio´n, Servicio de Histologı´a, Hospital Ramo´n y Cajal, E-28034 Madrid, Spain b Departamento de Quı´mica de Proteı´nas, CSIC, E-28006 Madrid, Spain Received 15 July 1998; received in revised form 6 August 1998; accepted 12 August 1998
Abstract The central neuroprotective effects against ischemic injury of fibroblast growth factor (FGF), administered either directly into the central nervous system or systemically, is well documented. Here we show in a rat model of transient retinal ischemia that the neuroprotective effect of systemically administered acidic fibroblast growth factor (aFGF, FGF-1) extends to the retina. Histological findings show a lower decrease of retinal ganglion cells and inner nuclear layer cells (P , 0.0001) in animals receiving FGF-1. These results suggest that FGF may function as a natural protection agent during transient retinal ischemia and further document that an efficient neuroprotection of central nervous tissues can be obtained by systemic administration of this protein. Our data may, thus, contribute to the development of novel and safe therapeutic approach for the treatment of the ischemic injury of the retina. 1998 Elsevier Science Ireland Ltd. All rights reserved
Keywords: Retinal ischemia; Rat; Fibroblast growth factor; Neuroprotection
Fibroblast growth factors (FGFs) have been shown to boost brain recovery after ischemia. The rationale for this effect was suggested by the fact that the levels of these proteins significantly increase following ischemic injury [19], which has been interpreted to reflect a physiological protection mechanism, since FGFs are powerful survival promoters for several neuronal populations [2,10,17,21]. The inability of these proteins to cross the blood–brain barrier (BBB) has been assumed to be due to their relatively high molecular mass. Consequently, it has been proposed that their administration to the central nervous system (CNS) requires invasive procedures which, obviously, constitute a serious limitation for a widespread clinical application. However, recently reported data suggest that FGFs cross the BBB [3] at an adequate level for protecting brain tissues from ischemic-reperfusion injury [4,5,11]. Recently, the enhancement of the expression of FGF and its receptor mRNAs has been reported in ischemic-reper* Corresponding author. Tel./fax: +34 91 3368290; e-mail:[email protected]
fused rat retina [16], data that have been interpreted as a physiological self-protective mechanism for minimizing tissue damage. Indeed, the intracameral administration of FGF preserves inner retinal layers from ischemia [20,23]. As we have shown that systemically injected FGF readily accumulates at the retina [3], a series of studies, reported here, aimed at establishing whether intravenously administered acidic fibroblast growth factor (FGF-1) might ameliorate the severity of retinal injury caused by transient ischemic episodes, were undertaken. The experiments were performed in accordance to the guidelines summarized in [24]. Retinal ischemia and reperfusion were induced according to the technique described by Stefansson et al. [18]. In brief, Sprague–Dawley rats (250–300 g) of either sex were anesthetized with an intraperitoneal injection of 3ml/kg of a mixture of ketamine hydrochloride (2.5 mg/ml), valium (2 mg/ml) and atropine (0.1 mg/ml). After a lateral conjunctival peritomy and desinsertion of the lateral rectus muscle, the optic nerve of the right eye was exposed. A 6–0 nylon suture was passed behind the optic nerve and was tightened until blood flow ceased in all retinal vessels.
0304-3940/98/$19.00 1998 Elsevier Science Ireland Ltd. All rights reserved PII S0304- 3940(98) 00672- 7
2
P. Cuevas et al. / Neuroscience Letters 255 (1998) 1–4
The complete absence of blood flow was assessed with an operating microscope with a flat contact lens. Ninety minutes after the ischemia onset, the suture was removed, and reperfusion of the retinal vessels monitored through the operating microscope. Eyes that failed to reperfuse within 5 min were excluded from this experiment. Recombinant human FGF-1 was synthesized according to method of Zazo et al. [22] and dissolved in PBS containing 0.1% heparin. A volume of 2.6 mg aFGF was injected in 50 ml solution through a jugular vein at the onset of reperfusion (n = 10). The dosage and time of FGF administration were selected on the basis of our previous demonstration that such a dose protects the rat brain during postischemic reperfusion [4]. Control animals (n = 10) received 50 ml of vehicle solution in the same manner. Sham-operated animals (n = 5) were manipulated in a similar fashion without retinal vessels occlusion. Two days after the ischemic episode the animals were reanesthetized and retrogradely perfused through a heparinized cannula introduced into the abdominal aorta via the femoral artery. For washing out the blood through the excised inferior vena cava, 200 ml PBS containing 0.1% heparin was used. Fixation was performed with 200 ml of 4% formaldehyde. Then the ischemic and nonischemic eyes were enucleated and divided into two halves by coronal section through the ora serrata. The vitreous was removed, and the posterior half of the eye was immersed in the same fixative for 24 h. Specimens were dehydrated and embedded in paraffin. Horizontal sections (4 mm thickness) were stained with hematoxylin and eosin. Ischemic damage was quantified in two ways. First, the thickness of the outer nuclear and outer plexiform layers (ONL and OPL) were measured in three different sections at 1 mm from both sides of the optic nerve and compared with values to that in the contralateral nonischemic eye. Second, in the same sections the number of cells in ganglion
cell layer (GCL) and inner nuclear layer (INL) were counted from one ora serrata to the other ora serrata and compared with GCL and INL cell counts in contralateral nonischemic eye. The changes in retinal layer thickness and the number of cells were expressed in percent ratios with respect to those of the nonischemic eye. The statistical significance was subsequently evaluated by ANOVA and Student’s ttests. Histopathological changes after transient eye ischemia were limited mostly to the inner part of the retina; only small alterations were seen in the photoreceptor layer using light microscopy. Fig. 1 shows the changes in retinal layers of the ischemic vehicle eye compared to FGF-1 ischemic eye. Quantification of those differences appears summarized in Fig. 2. According to these data, the inner plexiform layer (IPL) thickness of the ischemic eyes, which received vehicle solution, and those treated with FGF-1 were (mean ± SEM) 52.4 ± 5,75 and 82.7 ± 5.04% of the nonischemic ones, respectively. These differences are significant at the level of P , 0.0003 (Fig. 2A). There were no significant differences in eye layer thickness for the ONL, OPL and IPL between the ischemic and non ischemic eyes. As shown in Fig. 2B, the percentage of surviving neurons in GCL is significantly lower in vehicle-treated ischemic eyes than in FGF-1-treated ones (26.5 ± 2.89 vs. 61.0 ± 3.29%, P , 0.0001). Finally, the ratios of nuclei cell counts in INL between the ischemic and nonischemic eyes was higher in the FGF-1-treated group than in that which received vehicle (80.30 ± 3.67 vs. 45.3 ± 2.94%, P , 0.0001) (Fig. 2C). No morphological disturbances have been observed in sham-operated retina. Severe edema is observed in vehicle-treated animals (Fig. 1). This finding could explain that INL thickness is similar in vehicle- and FGF-1-treated animals although in the INL from ischemic vehicle retina the number of cells is lower than in INL from FGF-1-treated retina. In addition, vehicle-
Fig. 1. Light micrographs of the posterior ischemic rat retinas. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; ONL, outer nuclear layer; R and C, layer of rods and cones. An eye treated with vehicle solution at the onset of reperfusion shows an inner retina that is reduced in thickness compared to an FGF-1-treated eye. Note that the vehicle-treated retina shows an intense edema. Hematoxylin and eosin staining (original magnification ×50).
P. Cuevas et al. / Neuroscience Letters 255 (1998) 1–4
3
Fig. 2. Statistic assessment of the effect of systemic administration of F-1 on the histological structure of the retina after a transient ischemia episode. (A) Mean thickness of ischemic retinal IPL as a percentage of the mean thickness of retinal IPL of the nonischemic contralateral eye. The mean thickness of IPL was significantly greater in the FGF-1 than in the vehicle-treated eyes (P , 0.0003). (B) Ischemic retinal ganglion cell number as a percentage of retinal ganglion cells of the nonischemic contralateral eye. Retinal ganglion cell counts were significantly higher in the FGF-1-treated eyes than in vehicle-treated ones (P , 0.0001). (C) Cell counts in ischemic INL as a percentage of cell counts in nonischemic contralateral INL. The difference between the vehicle group and the group receiving FGF-1 was significant (P , 0.0001).
treated eyes show inflammatory signs in the inner retinal layers. The absence of inflammatory responses following transient retinal ischemia observed in FGF-1-treated eyes could be explained by the postulated anti-inflammatory activities of FGF [13]. This report shows that transient retinal ischemia in the rat eye causes a decrease in the IPL thickness and supports that this decrease is a reliable parameter for ischemic loss of retinal ganglion cells [8]. The results of the present study confirm and extend observations that have indicated that intracameral administration of FGF preserves the inner retinal layers from ischemia [20,23]. Although the nature of the protective effect of FGF against retinal injury and its relation to protection from cell death remains to be investigated, it has been shown that FGF protects against neurotoxicity during brain ischemia by a buffering Ca2 + mechanism [6,7,15]. One possible mechanism whereby FGF protects retina from reperfusion injury may be mediated by the capacity of FGF to suppress the expression of NMDA receptor protein [14]. Since retinal ischemia involves NMDA receptor activation [1], suppression of NMDA receptors can stabilize Ca2+ and thereby protect neurons against degeneration. It has been shown that intraocular FGF administration protects retinal tissue against ischemia [20,23]. However, this method of FGF delivery is not without potential side effects [12]. In this report we show retinal neuroprotection after transient ischemia using a single systemic injection of FGF. In addition, this finding adds further arguments in favor of FGF penetration across BBB and is in accordance with other studies showing neuroprotective effects of peripherally delivered FGF [9,11]. We thank Ch. Bourdier for editorial assistance and A. Ferna´ndez for technical aid. This work was supported by Fundacio´n Futuro. [1] Bresnick, G.H., Excitotoxins: a possible new mechanism for the
[2]
[3]
[4]
[5]
[6]
[7]
[8] [9]
[10]
[11]
[12]
pathogenesis of ischemic retinal damage, Arch. Ophthalmol., 107 (1989) 339–341. Cheng, B. and Mattson, M.P., NGF and bFGF protect rat hippocampal and human cortical neurons against hypoglucemic damage by stabilising calcium homeostasis, Neuron, 7 (1991) 1031–1041. Cuevas, P., Fernandez Ayerdi, A., Carceller, F., Collin, S., Mascarelli, F., Mun˜oz Willery, Y. and Gime´nez-Gallego, G., Central nervous system distribution of fibroblast growth factor injected into the blood stream, Neurol. Res., 18 (1996) 267– 272. Cuevas, P., Carceller, F. and Gime´nez-Gallego, G., Intravenous fibroblast growth factor penetrates the blood–brain barrier and protects hippocampal neurons against ischemia-reperfusion injury, Surg. Neurol., 49 (1998) 77–84. Cuevas, P., Carceller, F., Reimers, D., Saenz de Tejada, Y. and Gime´nez-Gallego, G., Acidic fibroblast growth factor rescues gerbil hippocampal neurons from ischemic apoptotic death, Neurol. Res., 20 (1998) 271–274. Freese, A., Finklestein, S.P. and DiFiglia, M., Basic fibroblast growth factor protects striatal neurons in vitro from NMDAreceptor mediated excitotoxicity, Brain Res., 575 (1992) 351– 355. Heidinger, V., Hicks, D., Sahel, J. and Dreyfus, H., Peptide growth factors but not ganglioside protect against excitotoxicity in rat retinal neurons in vitro, Brain Res., 767 (1997) 279– 288. Hughes, W.F., Quantitation of ischemic damage in the rat retina, Exp. Eye Res., 53 (1991) 573–582. Ikeda, K., Iwasaki, Y., Tagaya, N., Shiojima, T., Kobayashi, T. and Kinoshita, M., Neuroprotective effect of basic fibroblast growth factor on wobbler mouse motor neuron disease, Neurol. Res., 17 (1995) 445–448. Ishihara, A., Saito, H. and Nishiyama, N., Basic fibroblast growth factor ameliorates learning deficits in basal forebrainlesioned mice, Jpn. J. Pharmacol., 59 (1992) 7–13. Kirschner, P.B., Henshaw, R., Weise, J., Trubetskoy, V., Finklestein, S., Schultz, J.B. and Beal, M.F., Basic fibroblast growth factor protects against excitotoxicity and chemical hypoxia in both neonatal and adult rats, J. Cereb. Blood Flow Metab., 15 (1995) 619–623. Lavail, N.M., Unoki, K., Yasumura, D., Matthes, M.T., Yancopoulos, G.D. and Steinberg, R.H., Multiple growth factors, cytokines and neurotrophins rescue photoreceptors from
4
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P. Cuevas et al. / Neuroscience Letters 255 (1998) 1–4 the damaging effects of constant light, Proc. Natl. Acad. Sci. USA, 89 (1992) 11249–11253. Lefaucher, J.P., Gjata, B., Lafont, H. and Sebile, A., Angiogenic and inflammatory responses following skeletal muscle injury are altered by immune neutralization of endogenous basic fibroblast growth factor insulin-like growth factor-1 and transforming growth factor-b1, J. Neuroimmunol., 70 (1996) 37–44. Matson, M.P., Kumar, K., Cheng, B., Wang, H. and Michelis, E.K., Basic FGF regulates the expression of a functional 71 Kda NMDA receptor protein that mediates calcium influx and neurotoxicity in cultured hippocampal neurons, J. Neurosci., 13 (1993) 4575–4588. Mattson, M.P., Cheng, B. and Smith-Swintosky, V.L., Growth factor-mediated protection from excitotoxicity and disturbances in calcium and free radical metabolism, Semin. Neurosci., 5 (1993) 295–307. Miyashiro, M., Ogata, N., Takahashi, Kl., Matsushima, M., Yamamoto, C., Yamada, H. and Uyama, M., Expression of basic fibroblast growth factor and its receptor mRNA in retinal tissue following ischemic injury in the rat, Graefe’s Arch. Clin. Exp. Ophthalmol., 236 (1998) 295–300. Otto, D. and Unsicker, K., Basic FGF reverses chemical and morphological deficits in the nigrostriatal system of MPTP-treated mice, J. Neurosci., 10 (1990) 1912–1921. Stefansson, E., Wilson, C.A., Schoen, T. and Kuwabara, T.,
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