Mild stress culture conditions promote neurite outgrowth of retinal explants from postnatal mice

Highlights

• Short-term osmotic stress and heat stress can enhance RGC neurite outgrowth from P9-P11 mouse explants.

• The effect of mild stress (osmotic and heat) on RGC neurite outgrowth is largely a result of increased neural activity.

• Expression of heat shock proteins 27 and 70 is correlated with the mild heat stress in promoting RGC neurite outgrowth.

• These results provide significant insights into the development of therapeutic strategy for axon regeneration of RGCs.

Abstract

The axons of retinal ganglion cells (RGCs) in adult mammals fail to regenerate after injury. It has been suggested that some extrinsic factors, such as neural activity, may promote the regeneration process. The present study tested the hypothesis that environmental stress such as slightly elevated osmolarity and temperature can enhance neural activity and thus promote axon regeneration of RGCs in postnatal mice. Retinal explants from P9-11 mice were cultured for 5 days to study the capacity of RGC neurite outgrowth. The neural activity of retinal explants in these two stress conditions was examined using the multi-electrode array. We found that RGC neurite outgrowth from P9-P11 mouse explants was significantly enhanced when the concentration of the culture medium was increased by 1.25 fold, but not when increased by 1.5 fold. Similarly, retinal explants from P9-P11 mice grew longer neurites when the overall temperature was increased from 35 °C to 38 °C, 40 °C or 42 °C for one hour each day, but not when they were kept at 40 °C or 42 °C constantly for five days. We further showed that there was increased neural activity during these two mild stress conditions. It was found that short-term 42 °C heat stress increased the expression of heat shock proteins 27 and 70 in postnatal retinas and they were RGC neural activity dependent. The present study thus provides insights into the cellular mechanism of retinal axon regeneration under the mild stress conditions.

Introduction

Failed axon regeneration within the central nervous system (CNS) after injury is mainly caused by lacking of extrinsic regenerative environment, such as the formation of a glia scar (Fitch and Silver, 2008) and the presence of inhibitory factors (Cajal, 1928, Liu et al., 2011). In this context, retinal ganglion cells (RGCs), a type of CNS neuron that process a range of visual information and project this information from the eye to various areas of the brain (van Essen et al., 1992), are unable to regenerate their axons after injury. Glaucoma is the most common optic neuropathy and is characterized by RGC axon degeneration that eventually results in visual field loss and irreversible blindness. Thus, finding the ways of promoting RGC axon regeneration is critical to improving the treatment of glaucoma and other optic nerve injuries.

In a range of different nervous systems (both PNS and CNS), it has been shown that short-term electrical stimulation is able to regulate axon growth (Gordon et al., 2009, Hamid and Hayek, 2008). In RGCs, it is well known that neural activity plays a critical role in mediating neuronal survival and axon regeneration (Corredor and Goldberg, 2009). In addition, it has been demonstrated that electrical activation is able to enhance both the survival and the axon growth of cultured RGCs in response to brain-derived neurotrophic factor (BDNF) in rats (Goldberg et al., 2002). In a recent study, it was reported that short-term electrical stimulation also enhances the neurite outgrowth of retinal explants from postnatal mice (Lee and Chiao, 2016). Furthermore, a combination of increased neural activity by visual stimulation and activation of the mTOR signaling pathway has been shown to significantly promote long-distance adult retinal axon regeneration in vivo (Lim et al., 2016). Taken together, it is well established that neural activity is an important factor when attempting to develop effective therapies for RGC survival and axon regeneration after injury and glaucoma.

Stress is common in the natural world. At the cellular level, hyperthermia, hypothermia, an altered pH, oxygen deprivation, and situations involving a highly oxidative environment are all stresses that interfere directly and indirectly with protein functioning and the signaling pathways in cells. For example, in plants, heat and drought stresses result in the closure of stomata, a suppression of photosynthesis, and an increase in leaf temperature (Rizhsky et al., 2002). In mammals, most cells appear to be incapable of adapting to higher temperatures and synthesize harmful stress proteins when the environmental temperature increases (Welch, 1992). However, when confronted with mild stress, cells are able to initiate protective signaling pathways that allow adaption to the adverse environment and thus promote cell survival. For example, a transient heat shock pretreatment has been shown to protect against seizure and epilepsy-induced cell damage in rats (Duveau et al., 2005). In the retina, it has also been reported that local hypothermia greatly protects the retina from 120 min of ischemic injury (Sloane, 1989). Thus, it seems that mild stress, but not high levels of stress, is beneficial in terms of cell survival and protection of the cellular environment.

In a serendipitous finding, we found that a slight increase in culture medium concentration as a result of accidental elevation of the temperature of a 12-well plate by the heat from the rotator below and resulted in partial evaporation of the culture medium produced enhanced neurite outgrowth of retinal explants using a P11 mouse model (Fig. 1B and C). It should be noted that the culture medium lacked BDNF or IGF-1, which suggests that the mild stress due to the medium's increased osmolarity may contribute to the observed increase in neurite outgrowth. In addition to osmotic pressure, heat is also a common stress that affects cell survival and growth. Heat shock proteins 27 and 70 (HSP27 and HSP70) are expressed in the retina after trauma (Chidlow et al., 2014, Costigan et al., 1998, Hebb et al., 2006, Krueger-Naug et al., 2002, Liu et al., 2013), and they are known to play a crucial role in neuronal survival and neurite outgrowth (Hirata et al., 2003, Ousman et al., 2017). In a recent study, it was shown that expression of HSP70 is able to improve RGC cell survival after stress and damage (Piri et al., 2016). It has also been reported that the expression of intact HSP70 is likely to protect photoreceptor cells during retinal degeneration (Furukawa and Koriyama, 2016), and adeno-associated virus-2 (AAV2)-mediated induction of HSP70 expression is known to increase RGC survival after axonal injury (Kwong et al., 2015). Furthermore, it has been found that HSP27 is able to promote survival and axonal growth after adult peripheral nerve injury (Costigan et al., 1998, Williams et al., 2006). In addition, expression of HSP27 is increased in parallel with axonal regeneration of mature RGCs (Hebb et al., 2006). Taking all of the above together, it would seem that mild stress is an important factor that affects cell survival in the nervous system and it can also influence axon regeneration in such nervous systems. In the present study, we examined the effect of stress induced by osmolality and heat on neurite outgrowth of P9-P11 retinal explants, and it was found that mild stress is able to promote neurite outgrowth by increasing neural activity and upregulating expression of HSPs.