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Activation of Neuropeptide Y Receptors Modulates Retinal Ganglion Cell Physiology and Exerts Neuroprotective Actions In Vitro.

Martins J, Elvas F, Brudzewsky D, Martins T, Kolomiets B, Tralhão P, Gøtzsche CR, Cavadas C, Castelo-Branco M, Woldbye DP, Picaud S, Santiago AR, Ambrósio AF - ASN Neuro (2015)

Bottom Line: We found that NPY attenuated the increase in the [Ca2+]i triggered by glutamate mainly via Y1 receptor activation.Using in vitro cultures of rat retinal explants exposed to NMDA, we found that NPY pretreatment prevented NMDA-induced cell death.In conclusion, we found modulatory effects of NPY application that for the first time were detected at the level of RGCs.

View Article: PubMed Central - PubMed

Affiliation: Institute for Biomedical Imaging and Life Sciences (IBILI), Faculty of Medicine, University of Coimbra, 3004-548 Coimbra, Portugal CNC.IBILI, University of Coimbra, 3004-548 Coimbra, Portugal.

No MeSH data available.


Related in: MedlinePlus

Intravitreal administration of NPY or (Leu31, Pro34) − NPY did not prevent cell death induced by retinal I-R injury at 24 h of reperfusion. Retinal ischemia was induced for 60 min followed by 24 h of reperfusion. Ischemia was induced in one eye, and the contralateral eye was taken as an internal control. (a) Representative images of retinal sections showing TUNEL-positive cells (green) and Brn3a-positive RGCs (red). Nuclei were stained with DAPI (blue). Saline (aa, ad), 10 µg (2.34 nmol) NPY (ab, ae), or 10 µg (2.36 nmol; Leu31, Pro34) − NPY (ac, af) were intravitreally injected 2 h before the onset of I-R injury. GCL = ganglion cell layer; IPL = inner plexiform layer; INL = inner nuclear layer; OPL = outer plexiform layer; ONL = outer nuclear layer. Scale bar: 50 µm. (b) TUNEL-positive cells are Figure 7. Continued. expressed per mm of section length. I-R injury induced an increase in the number of TUNEL-positive cells. NPY or (Leu31, Pro34) − NPY administration was not able to reduce the number of TUNEL-positive cells. (c) Brn3a-positive RGCs were expressed per mm of section length. NPY or (Leu31, Pro34) − NPY administration was not able to prevent the reduction in Brn3a-positive RGC number induced by I-R injury. (d) Examples of scotopic ERG traces for saline (da, dd), NPY-treated (db, de), or (Leu31, Pro34) − NPY-treated eyes (dc, df). ERG recordings were performed before (baseline) and 24 h after I-R injury. Note that in the injured eye the b-wave was reduced (dd). NPY or (Leu31, Pro34) − NPY administration did not prevent the reduction in b-wave (de, df). Data are presented as mean ± SEM of n = 5 to 6 independent experiments. *p < .05, **p < .01, compared with contralateral eye. Kruskal-Wallis followed by Dunn’s test.
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fig7-1759091415598292: Intravitreal administration of NPY or (Leu31, Pro34) − NPY did not prevent cell death induced by retinal I-R injury at 24 h of reperfusion. Retinal ischemia was induced for 60 min followed by 24 h of reperfusion. Ischemia was induced in one eye, and the contralateral eye was taken as an internal control. (a) Representative images of retinal sections showing TUNEL-positive cells (green) and Brn3a-positive RGCs (red). Nuclei were stained with DAPI (blue). Saline (aa, ad), 10 µg (2.34 nmol) NPY (ab, ae), or 10 µg (2.36 nmol; Leu31, Pro34) − NPY (ac, af) were intravitreally injected 2 h before the onset of I-R injury. GCL = ganglion cell layer; IPL = inner plexiform layer; INL = inner nuclear layer; OPL = outer plexiform layer; ONL = outer nuclear layer. Scale bar: 50 µm. (b) TUNEL-positive cells are Figure 7. Continued. expressed per mm of section length. I-R injury induced an increase in the number of TUNEL-positive cells. NPY or (Leu31, Pro34) − NPY administration was not able to reduce the number of TUNEL-positive cells. (c) Brn3a-positive RGCs were expressed per mm of section length. NPY or (Leu31, Pro34) − NPY administration was not able to prevent the reduction in Brn3a-positive RGC number induced by I-R injury. (d) Examples of scotopic ERG traces for saline (da, dd), NPY-treated (db, de), or (Leu31, Pro34) − NPY-treated eyes (dc, df). ERG recordings were performed before (baseline) and 24 h after I-R injury. Note that in the injured eye the b-wave was reduced (dd). NPY or (Leu31, Pro34) − NPY administration did not prevent the reduction in b-wave (de, df). Data are presented as mean ± SEM of n = 5 to 6 independent experiments. *p < .05, **p < .01, compared with contralateral eye. Kruskal-Wallis followed by Dunn’s test.

Mentions: In a previous study from our group, pretreatment with NPY before the intravitreal injection of glutamate was shown to afford neuroprotection to RGCs (Santos-Carvalho et al., 2013a). Here, we explored the potential neuroprotective action of NPY in an animal model of retinal damage induced by I-R injury (Figure 7). Saline (n = 5 animals), 10 µg (2.34 nmol) NPY (n = 6), or 10 µg (2.36 nmol; Leu31, Pro34) − NPY (Y1/Y5 receptor agonist, n = 5) were intravitreally injected 2 h prior to I-R injury. Retinal cell death was assessed by TUNEL assay. I-R injury induced an increase in the number of TUNEL-positive cells (31.8 ± 6.6) per mm of retinal section length across all the retinal nuclear layers compared with the retina of the contralateral eye (0.6 ± 0.3 TUNEL-positive cells; Figure 7(b)). This increase was not prevented by pretreatment with NPY or (Leu31, Pro34) − NPY. RGC survival was evaluated by counting the number of Brn3a-immunoreactive cells, a RGC marker Brn3a (Nadal-Nicolas et al., 2012). We found that I-R injury decreased the number of Brn3a-positive RGCs per mm of retinal section length to 12.0 ± 2.5 RGCs compared with the contralateral eye (26.0 ± 4.5 Brn3a-positive cells), and again, this effect was not prevented by NPY or (Leu31, Pro34) − NPY administration (Figure 7(c)). We also performed ERGs in order to assess the functional effect of I-R injury on retinal light responses and a potential protective effect of NPY. Scotopic and photopic ERGs were recorded before the onset of I-R injury (baseline) and after 24 h of reperfusion (Figure 7(d)). We found that I-R injury induced a clear decrease in scotopic and photopic b-wave amplitudes but not in scotopic a-wave amplitude (data not shown). Similarly to results on TUNEL and Brn3a assays, the NPY or (Leu31, Pro34) − NPY administration before I-R injury did not prevent the reduction in ERG b-wave amplitudes. Together, these results indicate that, contrarily to the protective action against an excitotoxic injury in retinal explants, NPY or (Leu31, Pro34) − NPY pretreatment was not able to prevent retinal I-R injury within 24 h of reperfusion.Figure 7.


Activation of Neuropeptide Y Receptors Modulates Retinal Ganglion Cell Physiology and Exerts Neuroprotective Actions In Vitro.

Martins J, Elvas F, Brudzewsky D, Martins T, Kolomiets B, Tralhão P, Gøtzsche CR, Cavadas C, Castelo-Branco M, Woldbye DP, Picaud S, Santiago AR, Ambrósio AF - ASN Neuro (2015)

Intravitreal administration of NPY or (Leu31, Pro34) − NPY did not prevent cell death induced by retinal I-R injury at 24 h of reperfusion. Retinal ischemia was induced for 60 min followed by 24 h of reperfusion. Ischemia was induced in one eye, and the contralateral eye was taken as an internal control. (a) Representative images of retinal sections showing TUNEL-positive cells (green) and Brn3a-positive RGCs (red). Nuclei were stained with DAPI (blue). Saline (aa, ad), 10 µg (2.34 nmol) NPY (ab, ae), or 10 µg (2.36 nmol; Leu31, Pro34) − NPY (ac, af) were intravitreally injected 2 h before the onset of I-R injury. GCL = ganglion cell layer; IPL = inner plexiform layer; INL = inner nuclear layer; OPL = outer plexiform layer; ONL = outer nuclear layer. Scale bar: 50 µm. (b) TUNEL-positive cells are Figure 7. Continued. expressed per mm of section length. I-R injury induced an increase in the number of TUNEL-positive cells. NPY or (Leu31, Pro34) − NPY administration was not able to reduce the number of TUNEL-positive cells. (c) Brn3a-positive RGCs were expressed per mm of section length. NPY or (Leu31, Pro34) − NPY administration was not able to prevent the reduction in Brn3a-positive RGC number induced by I-R injury. (d) Examples of scotopic ERG traces for saline (da, dd), NPY-treated (db, de), or (Leu31, Pro34) − NPY-treated eyes (dc, df). ERG recordings were performed before (baseline) and 24 h after I-R injury. Note that in the injured eye the b-wave was reduced (dd). NPY or (Leu31, Pro34) − NPY administration did not prevent the reduction in b-wave (de, df). Data are presented as mean ± SEM of n = 5 to 6 independent experiments. *p < .05, **p < .01, compared with contralateral eye. Kruskal-Wallis followed by Dunn’s test.
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fig7-1759091415598292: Intravitreal administration of NPY or (Leu31, Pro34) − NPY did not prevent cell death induced by retinal I-R injury at 24 h of reperfusion. Retinal ischemia was induced for 60 min followed by 24 h of reperfusion. Ischemia was induced in one eye, and the contralateral eye was taken as an internal control. (a) Representative images of retinal sections showing TUNEL-positive cells (green) and Brn3a-positive RGCs (red). Nuclei were stained with DAPI (blue). Saline (aa, ad), 10 µg (2.34 nmol) NPY (ab, ae), or 10 µg (2.36 nmol; Leu31, Pro34) − NPY (ac, af) were intravitreally injected 2 h before the onset of I-R injury. GCL = ganglion cell layer; IPL = inner plexiform layer; INL = inner nuclear layer; OPL = outer plexiform layer; ONL = outer nuclear layer. Scale bar: 50 µm. (b) TUNEL-positive cells are Figure 7. Continued. expressed per mm of section length. I-R injury induced an increase in the number of TUNEL-positive cells. NPY or (Leu31, Pro34) − NPY administration was not able to reduce the number of TUNEL-positive cells. (c) Brn3a-positive RGCs were expressed per mm of section length. NPY or (Leu31, Pro34) − NPY administration was not able to prevent the reduction in Brn3a-positive RGC number induced by I-R injury. (d) Examples of scotopic ERG traces for saline (da, dd), NPY-treated (db, de), or (Leu31, Pro34) − NPY-treated eyes (dc, df). ERG recordings were performed before (baseline) and 24 h after I-R injury. Note that in the injured eye the b-wave was reduced (dd). NPY or (Leu31, Pro34) − NPY administration did not prevent the reduction in b-wave (de, df). Data are presented as mean ± SEM of n = 5 to 6 independent experiments. *p < .05, **p < .01, compared with contralateral eye. Kruskal-Wallis followed by Dunn’s test.
Mentions: In a previous study from our group, pretreatment with NPY before the intravitreal injection of glutamate was shown to afford neuroprotection to RGCs (Santos-Carvalho et al., 2013a). Here, we explored the potential neuroprotective action of NPY in an animal model of retinal damage induced by I-R injury (Figure 7). Saline (n = 5 animals), 10 µg (2.34 nmol) NPY (n = 6), or 10 µg (2.36 nmol; Leu31, Pro34) − NPY (Y1/Y5 receptor agonist, n = 5) were intravitreally injected 2 h prior to I-R injury. Retinal cell death was assessed by TUNEL assay. I-R injury induced an increase in the number of TUNEL-positive cells (31.8 ± 6.6) per mm of retinal section length across all the retinal nuclear layers compared with the retina of the contralateral eye (0.6 ± 0.3 TUNEL-positive cells; Figure 7(b)). This increase was not prevented by pretreatment with NPY or (Leu31, Pro34) − NPY. RGC survival was evaluated by counting the number of Brn3a-immunoreactive cells, a RGC marker Brn3a (Nadal-Nicolas et al., 2012). We found that I-R injury decreased the number of Brn3a-positive RGCs per mm of retinal section length to 12.0 ± 2.5 RGCs compared with the contralateral eye (26.0 ± 4.5 Brn3a-positive cells), and again, this effect was not prevented by NPY or (Leu31, Pro34) − NPY administration (Figure 7(c)). We also performed ERGs in order to assess the functional effect of I-R injury on retinal light responses and a potential protective effect of NPY. Scotopic and photopic ERGs were recorded before the onset of I-R injury (baseline) and after 24 h of reperfusion (Figure 7(d)). We found that I-R injury induced a clear decrease in scotopic and photopic b-wave amplitudes but not in scotopic a-wave amplitude (data not shown). Similarly to results on TUNEL and Brn3a assays, the NPY or (Leu31, Pro34) − NPY administration before I-R injury did not prevent the reduction in ERG b-wave amplitudes. Together, these results indicate that, contrarily to the protective action against an excitotoxic injury in retinal explants, NPY or (Leu31, Pro34) − NPY pretreatment was not able to prevent retinal I-R injury within 24 h of reperfusion.Figure 7.

Bottom Line: We found that NPY attenuated the increase in the [Ca2+]i triggered by glutamate mainly via Y1 receptor activation.Using in vitro cultures of rat retinal explants exposed to NMDA, we found that NPY pretreatment prevented NMDA-induced cell death.In conclusion, we found modulatory effects of NPY application that for the first time were detected at the level of RGCs.

View Article: PubMed Central - PubMed

Affiliation: Institute for Biomedical Imaging and Life Sciences (IBILI), Faculty of Medicine, University of Coimbra, 3004-548 Coimbra, Portugal CNC.IBILI, University of Coimbra, 3004-548 Coimbra, Portugal.

No MeSH data available.


Related in: MedlinePlus