Ketogenic diets have been used for over 80 years in the treatment of epilepsy. Recently, neuroprotective properties of ketone bodies have been found in diverse models of such neurodegenerative diseases of the brain as Parkinson’s or Alzheimer’s disease, and in models of brain trauma, hypoxia or ischemia [3
]. To date, however, similar studies regarding retinal degeneration have not been performed.
Excitotoxicity, which contributes to free radical formation and neuronal cell death, is an important factor in diverse chronic neurodegenerative diseases in the brain and also in the retina [4
]. Excitatory neurotoxicity is mediated in part by overactivation of the NMDA-type glutamate receptor [12
]. In the present study, an excitotoxic rat model of NMDA-induced RGC damage [24
] was used to evaluate the neuroprotective properties of systemic application of the ketone bodies ACA and BHB in the retina.
In rats, a single intravitreal NMDA injection reduced the RGC number to around 10% of that in PBS-injected controls. This RGC loss is comparable to that found in other studies using NMDA-induced retinal degeneration [25
However, ACA and BHB ameliorated the cell damage induced by NMDA, and resulted in a relative RGC increase of 48.4% and 41.5% respectively when administered intraperitoneally for 21 days.
It is possible that the lithium fraction in the lithium salt of ACA leads to neuroprotective effects, as shown previously in a rat model of partial optic nerve crush. However, the amount of lithium in doses of ACA as high as 250 mg/kg is less than that which showed a significant neuroprotective effect in previous studies of the retina [27
]. Furthermore, Massieu et al. demonstrated a protective effect of the lithium salt of ACA (250 mg/kg i.p.) but not of the lithium fraction alone in a model of glutamate-mediated neuronal damage in the hippocampus [4
]. Although an additive effect of lithium and ACA cannot be excluded, a substantial modification of ACA’s effects by the lithium fraction seems unlikely. Moreover, the neuroprotective effect of BHB against RGC death was comparable to that of ACA, even though BHB is not a lithium compound.
Two other studies found similar results for ACA but not for BHB in vivo, whereas the effective dose of intraperitoneal ACA after glutamate-mediated neuronal damage in the hippocampus was the same as in our study [4
], BHB at 332 mg/kg failed to achieve a neuroprotective effect [28
]. In contrast, another study on glutamate-mediated neuronal damage showed reduced levels of lipoperoxidation after a single intraperitoneal injection of 500 mg/kg BHB [29
], and Suzuki et al. demonstrated prolonged survival times after intravenous application of 30 mg/kg BHB in a rat model of brain anoxia [6
]. In all these studies, however, treatment times were shorter than in our study.
Plasma levels of ACA and BHB in rats have been shown to be increased from 0.06 mM (controls) to 0.6 mM 10 minutes after a single injection of 500 mg/kg ACA or BHB [29
]. However, in vitro considerably higher concentrations (5 mM ACA and 4 mM BHB) were the most effective against glutamate toxicity in a mouse hippocampal cell line [16
], in good agreement with our in vitro results in bovine retinal tissue.
How ketone bodies confer their neuroprotective effect on neuronal cells remains unclear, and several theories are currently under discussion. An important underlying mechanism may be an increase in neuronal stability due to enhanced ATP production [30
]. In accordance with this theory, Bough et al. found an increased number of mitochondrial profiles in rats fed a ketogenic diet, suggesting a stimulation of mitochondrial biogenesis and a higher phosphocreatine/creatine ratio in the hippocampus and higher available energy reserves [32
]. Such an increased energy level of the neurons might ensure them a better supply of the Na/K-ATPase, leading to enhanced membrane stability, and thus providing an improved resistance to metabolic stress.
Other studies have found protective effects of ketogenic diet or ketone bodies against excitotoxic neuronal degeneration, and proposed that oxidative stress modulation rather than glutamate receptor modulation may be important for this effect [4
]. Direct interaction of ketone bodies with glutamate receptors has been shown only for L-BHB (voltage-dependent blockade of NMDA receptors), whereas ACA or D-BHB were not able to reduce NMDA, AMPA or Kainate receptor-induced currents [34
]. Furthermore, even though ACA has been demonstrated to have a protective effect in hippocampal cells, this protective effect was also found in cells which lacked glutamate receptors [16
]. Such neuroprotective effects are probably due indirectly to an enhancement of cellular antioxidant capacity. Several studies describe an influence of ketone bodies on different antioxidative mechanisms of neuronal cells. Veech et al. showed that BHB is able to oxidize co-enzyme Q and reduce NADP+, thus reducing free radical formation [36
]. In addition, the reduction of mitochondrial free radical formation resulting from an increase the NAD+
/NADH ratio, along with the scavenging capacities of ACA and BHB for diverse reactive oxygen species (ROS), may contribute to neuroprotective effects, even when increase in oxidative stress is increased by excitotoxicity [29
Other factors which possibly play a role here include upregulation of glutathione peroxidase activity, an enzyme that can prevent lipid peroxidation, and upregulation of calbindin [33
In addition to an indirect influence on glutamate-induced neurotoxicity via the antioxidant effects of ketogenic diet or ketone bodies, indirect glutamate receptor modulation via reversal of the NMDA-inhibiting effect on the production of KYNA, an endogenous NMDA receptor modulator, has been postulated, and may play an additional role in preventing glutamate-induced neurotoxicity [19
]. In our study, NMDA-induced reduction of de novo KYNA production in ACA- and BHB-incubated bovine retinas in vitro was significantly attenuated. This effect was much more pronounced than the increase in RGC numbers after treatment with ketone bodies in vivo; therefore, a sole survival effect of KYNA-producing cells in the in vitro setting seems rather unlikely. The in vivo situation, however, is not directly comparable to the in vitro setting; further studies have to show in detail how ketone bodies modify KYNA production in the context of excitotoxicity. Modulation of NMDA receptors via alteration of KYNA-levels is especially of therapeutic interest. Since NMDA receptors are essential for neuronal transmission, a full blockade of NMDA receptors would not be suitable for clinical application; rather, it would be preferable to prevent receptor overstimulation while reducing free radical formation [41
]. Ketone bodies therefore seem to offer interesting features for treatment or prevention of cellular damage in different neuronal degenerations.
This study showed for the first time that systemically applied ketone bodies exert a partial neuroprotective action against NMDA-induced RGC damage in rats. Moreover, ACA and BHB were found to attenuate NMDA-induced inhibition of KYNA production in retinas in vitro. We believe that the use of ketone bodies or ketogenic diets should be considered and further evaluated in the quest for effective therapeutic approaches in the treatment of retinal degenerations.