Regenerative Effect of Growth Hormone (GH) in the Retina after Kainic Acid Excitotoxic Damage

Abstract

In addition to its role as an endocrine messenger, growth hormone (GH) also acts as a neurotrophic factor in the central nervous system (CNS), whose effects are involved in neuroprotection, axonal growth, and synaptogenic modulation. An increasing amount of clinical evidence shows a beneficial effect of GH treatment in patients with brain trauma, stroke, spinal cord injury, impaired cognitive function, and neurodegenerative processes. In response to injury, Müller cells transdifferentiate into neural progenitors and proliferate, which constitutes an early regenerative process in the chicken retina. In this work, we studied the long-term protective effect of GH after causing severe excitotoxic damage in the retina. Thus, an acute neural injury was induced via the intravitreal injection of kainic acid (KA, 20 µg), which was followed by chronic administration of GH (10 injections [300 ng] over 21 days). Damage provoked a severe disruption of several retinal layers. However, in KA-damaged retinas treated with GH, we observed a significant restoration of the inner plexiform layer (IPL, 2.4-fold) and inner nuclear layer (INL, 1.5-fold) thickness and a general improvement of the retinal structure. In addition, we also observed an increase in the expression of several genes involved in important regenerative pathways, including: synaptogenic markers (DLG1, NRXN1, GAP43); glutamate receptor subunits (NR1 and GRIK4); pro-survival factors (BDNF, Bcl-2 and TNF-R2); and Notch signaling proteins (Notch1 and Hes5). Interestingly, Müller cell transdifferentiation markers (Sox2 and FGF2) were upregulated by this long-term chronic GH treatment. These results are consistent with a significant increase in the number of BrdU-positive cells observed in the KA-damaged retina, which was induced by GH administration. Our data suggest that GH is able to facilitate the early proliferative response of the injured retina and enhance the regeneration of neurite interconnections.

Keywords: excitotoxicity; growth hormone; neurotrophic; regeneration; retina; synaptogenic.

Figure 1

Morphometric analysis showing the regenerative effect of growth hormone (GH) after kainic acid (KA)-induced excitotoxicity in chicken retinas. (A) Treatments and time-line schematic representation of intravitreal injection protocols in the experimental groups. Units in nanograms (ng) and micrograms (μg). P: postnatal day. (B) Histological analysis in hematoxylin-stained chicken retinal slices treated with either KA (damage), GH (treatment) or GH + KA (neuroprotection treatment) in the left eye. Sham (vehicle injected) as negative control (right eye). The thickness of retinal layers was quantified after each treatment: (C) from photoreceptors (PR) to ganglion cell layer (GCL); (D) inner plexiform layer (IPL) and (E) inner nuclear layer (INL). Bars indicate thickness mean ± SEM (n = 3–5 animals per group, 3 fields were quantified per retina/animal). Asterisks indicate significant difference in comparison to control (*, p < 0.05; ***, p < 0.001, ****, p < 0.0001) and number sign (#) shows difference between experimental groups (#, p < 0.05; ####, p < 0.0001) as determined by one-way ANOVA for multiple comparisons and Šidák as post-hoc test.

Figure 2

Effects of growth hormone (GH) upon DLG1, NRXN1, SNAP25, NR1, and GRIK mRNAs expression, and GAP43 immunoreactivity in chicken retinas exposed to excitotoxic damage. Panels show the relative expression of: (A) DLG1, (B) NRXN1, (C) SNAP25, (E) NR1, and (F) GRIK4 mRNAs, as determined by RT-qPCR. Relative mRNA expression values were corrected by the comparative threshold cycle (Ct) method and employing the formula 2^−∆∆CT. Ribosomal 18S RNA was used as housekeeping gene. (D) GAP43 immunoreactivity was analyzed by western blot and densitometry and normalized using β-actin as a loading control; 1, 2 and 3 represent different samples. A representative luminogram is shown. Bars represent mean ± SEM (n = 4–5; analyzed by duplicate). Asterisks indicate significant difference in comparison to control (*, p < 0.05; **, p < 0.01; ****, p < 0.0001) and number sign (#) shows difference between experimental groups (##, p < 0.01; ###, p < 0.001; ####, p < 0.0001) as determined by one-way ANOVA for multiple comparisons and Šidák as post-hoc test.

Figure 3

Effect of growth hormone (GH) upon Bcl-2 and TNF R2 immunoreactivity and BDNF, BMP4 and GH mRNAs expression in chicken retinas exposed to excitotoxic damage.(A) Representative western blot luminograms for Bcl-2 and TNF-R2 immunoreactivities in experimental groups. β-Actin was included as a loading and normalizing control. 1, 2 and 3 represent different samples. Immunoreactive bands were analyzed by densitometry for TNF-R2 (B) and Bcl-2 (C). Relative expression of: (D) BDNF, (E) BMP4, and (F) GH mRNAs, as determined by RT-qPCR. Relative mRNA expression values were corrected by the comparative threshold cycle (Ct) method and employing the formula 2^−∆∆CT. Ribosomal 18S RNA was used as housekeeping gene. Bars represent mean ± SEM (n = 4; analyzed by duplicate). Asterisks indicate significant difference in comparison to control (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001) and number sign (#) shows difference between experimental groups (#, p < 0.05; ####, p < 0.0001) as determined by one-way ANOVA for multiple comparisons and Šidák as post-hoc test.

Figure 4

Effect of growth hormone (GH) upon FGF2, Sox2, Ascl1, Notch1, Hes5 and PCNA mRNAs expression in chicken neuroretinas exposed to excitotoxic damage. Panels show the relative expression of (A) FGF2, (B) Sox2, (C) Acsl1, (D) Notch1, (E) Hes5, and (F) PCNA mRNAs, as determined by RT-qPCR. Relative mRNA expression values were corrected by the comparative threshold cycle (Ct) method and employing the formula 2^−∆∆CT. Ribosomal 18S RNA was used as housekeeping gene. Bars represent mean ± SEM (n = 4–5, analyzed by duplicate). Asterisks indicate significant difference in comparison to control (*, p < 0.05; **, p < 0.01; ***, p < 0.001) and number sign (#) shows difference between experimental groups (#, p < 0.05; ##, p < 0.01; ###, p < 0.001; ####, p < 0.0001) as determined by one-way ANOVA for multiple comparisons and Šidák as post-hoc test.

Figure 5

Proliferative effect of growth hormone (GH) treatment over BrdU immunoreactivity in the chicken retina exposed to excitotoxic damage. (A) Treatments and time-line schematic representation of intravitreal injection protocols in the experimental groups. Units in nanograms (ng), micrograms (μg) and milligrams (mg). P: postnatal day, I.V. intravitreal, I.P. intraperitoneal. (B) Positive BrdU cells, quantified as relative percentage in relation to DAPI labeled cells. BrdU immunofluorescence in control (sham), GH, kainic acid (KA) and KA + GH treated retinas. Sham (vehicle) and treated retinas were stained with DAPI (Blue; D, G, J, M, P), and with specific antibodies directed against BrdU (Green; C, F, I, L, O). Merged images are shown in panels E, H, K, N, and Q. Negative controls without primary antibody (O, P, and Q). Arrows denote co-localization of BrdU and DAPI in the same cells. Bars represent mean ± SEM (n = 3–5 animals per group, 3 fields were quantified per retina/animal). Asterisks indicate significant difference in comparison to control (****, P < 0.0001) and number sign (#) shows difference between experimental groups (#, P < 0.05) as determined by one-way ANOVA for multiple comparisons and LSD Fischer as post-hoc test.