(Discussion) Neuroprotection with Glycine-2-Methylproline-Glutamate (G-2MePE) after hypoxic-ischemic brain injury in adult rats

Discussion

In the present study G-2MePE treatment, by a single dose SC administration significantly reduced the extent of brain damage after HI injury. Attenuation of brain damage, neuronal loss and apoptosis were associated with reduced microglial reaction, TNF-α expression and greater astrocytosis in G-2MePE treated group.

The modified analogue of GPE, G-2MePE, has a much longer half-life in plasma (49 min) compared to GPE (2 min) due to improved enzymatic resistance (Bickerdike et al. , 2009). Although ANOVA indicated an overall reduction in tissue damage scores after single dose treatment the efficacy in individual brain regions was not significant. The treatment effects of G-2MePE were further also evaluated by measuring the number of survival neurons using NeuN immunolabelling. We found the significant improvements of neuronal survival in all brain regions examined. While the tissue damage scores reflected the changes that associated with necrotic neuronal death the evaluation of surviving neurons appeared to be a more sensitive measure for the treatment effects of G-2MePE. In addition, HI injury also induced obvious morphological changes in the neurons by showing the deformed and shrinking cell bodies. The treatment of G-2MePE also prevented HI-induced morphological changes in neurons.

The protective effects of G-2MePE were also demonstrated by showing reduced apoptotic neuronal death. It is known that the activation of caspase -3 is involved in neuronal death after hypoxic-ischemia particularly in brain regions with more progressive neuronal loss (Guan et al. , 2013). There were 50% more caspase-3 positive cells in the ipsilateral hippocampus than on the ipsilateral cortex, suggesting neuronal death may be more progressive in the hippocampus whereas necrotic death may be the major factor involved in cortical neuronal loss. Thus the protective effects of G-2MePE may be mediated through necrotic and apoptotic pathways.

Unilateral ligation of the carotid artery induces a relative ischemia without causing any tissue damage due to collateral blood supply from the contralateral hemisphere. The ischemia associated brain injury in the brain regions of middle cerebral artery territory occurs only when the collateral circulation is damaged, in this case the brief hypoxic insult. Unlike the ischemic-reperfusion injury from either a transient or permanent middle cerebral artery occlusion, the brain damage induced by HI appears to be more progressive and possibly more reversible. Consistent with the present study Bickerdike and colleagues (2009) reported that continuous intravenous infusion of G-2MePE reduces the infarct size after endothelin1 (ET1) induced middle cerebral artery vasoconstriction in rats (Bickerdike et al. , 2009). Zhao et al (2005) demonstrated that G-2MePE offers effective neuroprotection by reducing neuronal deficit scores at a 10 times lower dose compared to GPE, when treated in combination with caffeinol (Zhao et al. , 2005).

Microglia plays an important role in the inflammatory response in the brain and produce pro-inflammatory cytokines like TNF-α or IL-1β. We observed inhibition of reactive microglia and expression of TNF-α after G-2MePE treatment. This is consistent with evidence that anti-inflammation contributes to the neuroprotection of G-2MePE as the improved neuronal outcome was associated with reduction of pro-inflammatory cytokine IL-6 after HI injury in neonatal rats (Svedin et al. , 2007). The effects of TNF-α are mediated through two membrane receptors, TNF-α-receptor 1 &2, and activation of one of these receptors lead to cell death (Aggarwal and Natarajan, 1996, Nakazawa et al. , 2006).

The SC treatment with G-2MePE also induced a marked increase in reactive astrocytes in the ipsilateral brain after HI. Reactive astrocytes play a critical role in protecting neurons, maintaining blood-brain barrier integrity, cell-to-cell communication and neuronal plasticity (Kraig et al. , 1995). We have previously reported loss of astrocytes in the CA1-2 and CA4 sub-regions of the hippocampus after HI in adult rats and that treatment with GPE prevented the loss of astrocytes. This may be apparently associated with the increase in astrocytes in the present study (Guan, 2008). The effects of GPE on astrocytosis appeared to be brain region specific as there was no change in CA3 and the cortex. Astrocytes also have been implicated in the neo-vascularisation process (Salhia et al. , 2000, Acker et al. , 2001). Given a role for astrocytes in vascular remodelling, the astrocytosis may also be associated with the vascular protection and/or remodelling after GPE (Shapira et al. , 2009) and G-2MePE treatment in neonatal rats (Svedin et al. , 2007).

Given that GPE, presumably the analogue, does not interact with IGF-1 receptors the mechanism of neuroprotective effects of GPE has been recently suggested to be mediated through improving bioavailability of IGF-1 (Guan et al. , 2014), which needs to be further investigated in future study.

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