Neuroprotective and Regenerative Effects of Growth Hormone (GH) in the Embryonic Chicken Cerebral Pallium Exposed to Hypoxic-Ischemic (HI) Injury.

Prenatal hypoxic−ischemic (HI) injury inflicts severe damage on the developing brain provoked by a pathophysiological response that leads to neural structural lesions, synaptic loss, and neuronal death, which may result in a high risk of permanent neurological deficits or even newborn decease. It is known that growth hormone (GH) can act as a neurotrophic factor inducing neuroprotection, neurite growth, and synaptogenesis after HI injury. In this study we used the chicken embryo to develop both in vitro and in vivo models of prenatal HI injury in the cerebral pallium, which is the equivalent of brain cortex in mammals, to examine whether GH exerts neuroprotective and regenerative effects in this tissue and the putative mechanisms involved in these actions. For the in vitro experiments, pallial cell cultures obtained from chick embryos were incubated under HI conditions (<5% O2, 1 g/L glucose) for 24 h and treated with 10 nM GH, and then collected for analysis. For the in vivo experiments, chicken embryos (ED14) were injected in ovo with GH (2.25 µg), exposed to hypoxia (12% O2) for 6 h, and later the pallial tissue was obtained to perform the studies. Results show that GH exerted a clear anti-apoptotic effect and promoted cell survival and proliferation in HI-injured pallial neurons, in both in vitro and in vivo models. Neuroprotective actions of GH were associated with the activation of ERK1/2 and Bcl-2 signaling pathways. Remarkably, GH protected mature neurons that were particularly harmed by HI injury, but was also capable of stimulating neural precursors. In addition, GH stimulated restorative processes such as the number and length of neurite outgrowth and branching in HI-injured pallial neurons, and these effects were blocked by a specific GH antibody, thus indicating a direct action of GH. Furthermore, it was found that the local expression of several synaptogenic markers (NRXN1, NRXN3, GAP-43, and NLG1) and neurotrophic factors (GH, BDNF, NT-3, IGF-1, and BMP4) were increased after GH treatment during HI damage. Together, these results provide novel evidence supporting that GH exerts protective and restorative effects in brain pallium during prenatal HI injury, and these actions could be the result of a joint effect between GH and endogenous neurotrophic factors. Also, they encourage further research on the potential role of GH as a therapeutic complement in HI encephalopathy treatments.

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

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.

Gonadotropin-releasing hormone and growth hormone act as anti-inflammatory factors improving sensory recovery in female rats with thoracic spinal cord injury.

The potential for novel applications of classical hormones, such as gonadotropin-releasing hormone (GnRH) and growth hormone (GH), to counteract neural harm is based on their demonstrated neurotrophic effects in both in vitro and in vivo experimental models and a growing number of clinical trials. This study aimed to investigate the effects of chronic administration of GnRH and/or GH on the expression of several proinflammatory and glial activity markers in damaged neural tissues, as well as on sensory recovery, in animals submitted to thoracic spinal cord injury (SCI). Additionally, the effect of a combined GnRH + GH treatment was examined in comparison with single hormone administration. Spinal cord damage was induced by compression using catheter insufflation at thoracic vertebrae 10 (T10), resulting in significant motor and sensory deficits in the hindlimbs. Following SCI, treatments (GnRH, 60 µg/kg/12 h, IM; GH, 150 µg/kg/24 h, SC; the combination of both; or vehicle) were administered during either 3 or 5 weeks, beginning 24 h after injury onset and ending 24 h before sample collection. Our results indicate that a chronic treatment with GH and/or GnRH significantly reduced the expression of proinflammatory (IL6, IL1B, and iNOS) and glial activity (Iba1, CD86, CD206, vimentin, and GFAP) markers in the spinal cord tissue and improved sensory recovery in the lesioned animals. Furthermore, we found that the caudal section of the spinal cord was particularly responsive to GnRH or GH treatment, as well as to their combination. These findings provide evidence of an anti-inflammatory and glial-modulatory effect of GnRH and GH in an experimental model of SCI and suggest that these hormones can modulate the response of microglia, astrocytes, and infiltrated immune cells in the spinal cord tissue following injury.

Growth Hormone Neuroprotection Against Kainate Excitotoxicity in the Retina is Mediated by Notch/PTEN/Akt Signaling.

Purpose: In the retina, growth hormone (GH) promotes axonal growth, synaptic restoration, and protective actions against excitotoxicity. Notch signaling pathway is critical for neural development and participates in the retinal neuroregenerative process. We investigated the interaction of GH with Notch signaling pathway during its neuroprotective effect against excitotoxic damage in the chicken retina. Methods: Kainate (KA) was used as excitotoxic agent and changes in the mRNA expression of several signaling markers were determined by qPCR. Also, changes in phosphorylation and immunoreactivity were determined by Western blotting. Histology and immunohistochemistry were performed for morphometric analysis. Overexpression of GH was performed in the quail neuroretinal-derived immortalized cell line (QNR/D) cell line. Exogenous GH was administered to retinal primary cell cultures to study the activation of signaling pathways. Results: KA disrupted the retinal cytoarchitecture and induced significant cell loss in several retinal layers, but the coaddition of GH effectively prevented these adverse effects. We showed that GH upregulates the Notch signaling pathway during neuroprotection leading to phosphorylation of the PI3K/Akt signaling pathways through downregulation of PTEN. In contrast, cotreatment of GH with the Notch signaling inhibitor, DAPT, prevented its neuroprotective effect against KA. We identified binding sites in Notch1 and Notch2 genes for STAT5. Also, GH prevented Müller cell transdifferentiation and downregulated Sox2, FGF2, and PCNA after cotreatment with KA. Additionally, GH modified TNF receptors immunoreactivity suggesting anti-inflammatory actions. Conclusions: Our data indicate that the neuroprotective effects of GH against KA injury in the retina are mediated through the regulation of Notch signaling. Additionally, anti-inflammatory and antiproliferative effects were observed.