Short Communication Received: July 28, 2014 Accepted after revision: February 8, 2015 Published online: March 27, 2015
Ophthalmic Res 2015;53:162–167 DOI: 10.1159/000379753
Ketogenic Diet Attenuates NMDA-Induced Damage to Rat’s Retinal Ganglion Cells in an Age-Dependent Manner Tomasz Zarnowski a Tomasz J. Choragiewicz a, d Frank Schuettauf d Eberhart Zrenner d Robert Rejdak a, c, d Maciej Gasior e Iwona Zarnowska b Sebastian Thaler d
a Tadeusz Krwawicz Chair of Ophthalmology and Eye Hospital and b Department of Pediatric Neurology, Medical University, Lublin, and c Department of Experimental Pharmacology, PAS Medical Research Center, Warsaw, Poland; d Eye Hospital and Institute for Ophthalmic Research, Centre for Ophthalmology, University of Tübingen, Tübingen, Germany; e Department of Pharmacology and Physiology, Drexel University College of Medicine, Philadelphia, Pa., USA
Abstract Objective: This study was conducted to investigate neuroprotective effects of a high fat/low carbohydrate and protein diet (ketogenic diet, KD) in a model of N-methyl D-aspartate (NMDA)-induced retinal ganglion cell (RGC) damage in juvenile and young adult rats. Methods: Juvenile (30–35 days old) and young adult (56–70 days old) female Brown Norway rats were fed the KD for 21 days; rats exposed to a standard rodent diet (SRD) served as controls. The main constituents of the KD used in the present study were approximately 80% fats, 8% proteins, and less than 1% carbohydrates. On day 14 of exposure to the KD (or the SRD in the control group), each rat received a single intravitreal injection of NMDA; RGCs were then retrogradely labelled by hydroxystilbamidine on day 19 and collected on day 21 to assess the degree of damage induced by NMDA. Blood biomarkers to confirm the expected metabolic response to the KD (i.e. ketosis and hypo-
© 2015 S. Karger AG, Basel 0030–3747/15/0533–0162$39.50/0 E-Mail [email protected] www.karger.com/ore
glycaemia) were also assessed. Results: Although both the juvenile and young adult rats developed comparable ketosis and hypoglycaemia when fed the KD, NMDA-induced loss in RGCs was significantly attenuated only in juvenile rats exposed to the KD in comparison with those fed the SRD; exposure to the KD had no protective effect in young adult rats. In summary, exposure to the KD had a neuroprotective effect in NMDA-induced RGC damage in juvenile rats, but not in young adult rats. Conclusion: These results support further exploration of metabolic interventions to treat optic neuropathies associated with neurodegeneration. © 2015 S. Karger AG, Basel
Introduction
Glaucoma is a leading cause of irreversible blindness and is the most common optic neuropathy caused by the accelerated death of retinal ganglion cells (RGCs). A role of neurodegenerative mechanisms related to excitotoxicity, mitochondrial dysfunction, misfolding of various proteins, oxidative stress, and inflammation in RGC Maciej Gasior, MD, PhD Department of Pharmacology and Physiology Drexel University College of Medicine Philadelphia, PA (USA) E-Mail NextPharma @ gmail.com
Downloaded by: Univ. of California San Diego 132.239.1.231 - 6/8/2015 2:19:50 PM
Key Words Ketogenic diet · Neuroprotection · Retinal ganglion cells · Rat
next 21 days. The KD (F3666 Ketogenic Diet for Rodents; Bio-Serv, Frenchtown, N.J., USA) consisted of 78.8% fats, 8.4% proteins, 5% cellulose, less than 5% water, and less than 1% carbohydrates, and was supplemented with necessary minerals and amino acids (detailed information available at supplier’s http://www.bio-serv. com/pdf/F3666.pdf). The KD of this specific composition has been commonly used in pre-clinical research as its composition closely mimics that of the classic KD used clinically in patients [2, 3]. To confirm the development of ketosis and hypoglycaemia in rats fed the KD, blood concentrations of β-hydroxybutyrate and glucose were measured in rats on day 14 of exposure to the KD and in the control rats for comparison using the Precision Xtra Blood Glucose and Ketone Monitoring System (Abbott, Alameda, Calif., USA) [12]. The blood sample was collected by making a small incision on the skin of the tail. Assessing the degree of ketosis and hypoglycaemia served as a qualitative assurance that the expected metabolic changes due to exposure to the KD in the present study were comparable to those reported elsewhere. Concentrations of glucose (mg/dl) and β-hydroxybutyrate (mmol/l) are presented as group means ± SEM. Intravitreal Injections Intraperitoneal injections of chloral hydrate (7%, 6 ml/kg body weight) were used for anaesthesia. On day 14, each rat received a single intravitreal injection of NMDA (Sigma-Aldrich, St. Louis, Mo., USA) consisting of 2 μl of a 10 mmol/l NMDA solution in PBS using heat-pulled glass capillaries connected to a microsyringe (Drummond Scientific Co., Broomall, Pa., USA) under direct observation through the microscope. The NMDA dose was chosen in order to gain a reduction of healthy RGCs of about 90% in RGC counts of otherwise untreated animals, comparable to earlier studies [13]. This allowed studying potential neuroprotective effects of the KD under the worst scenario of nearly complete RCG damage. Rats in which lens damage occurred during this procedure were excluded from the study and were not used thereafter. In each case, the contralateral eye served as a control eye and was injected with PBS.
Diets Rats were fed ad libitum with a standard rodent diet (SRD, AIN-93G Basic Diet 2222 for Small Rodents; Kliba-Nafag, Kaiseraugst, Switzerland) before being divided into two groups. One group continued to be fed the SRD (control group) until the end of the experiment. The other group started to be fed the KD for the
Retrograde Labelling and Quantification of RGCs Five days after intraocular injection (day 19), the rats were anaesthetized, and a total of 7 μl of a fluorescing dye (hydroxystilbamidine methanesulfonate; Molecular Probes, Eugene, Oreg., USA) was applied to each superior colliculus with three stereotaxic-guided injections of 2.33 μl each at a depth of 3.5 mm (below brain surface). The injection site was a 1.5 × 2.5-mm rectangular window located between lambda and the bregma suture of the skull. On day 21 rats were sacrificed with CO2, the corneas of the treated eyes were marked for orientation in the form of retinal quadrants. The eyes were then enucleated, and the retinas were dissected, flat-mounted, and fixed in 2% paraformaldehyde. Visualization was performed immediately under a fluorescent microscope. Counting was carried out in 4 distinct areas (62,500 μm2 each) in the middle periphery of the retina corresponding to 2.0 mm distance from the optic nerve head at the 45, 135, 225, and 315° meridian, as described in detail previously [13]. Only one defined distance to the optic nerve was chosen in order to reduce animal numbers and increase sensitivity, due to the fact that RGC numbers are not uniformly distributed from the central to the peripheral regions of the rat retina [14]. Images were obtained using a digital imaging system connected to the microscope (ImagePro;
Ketogenic Diet and Retinal Ganglion Cell Damage in Rats
Ophthalmic Res 2015;53:162–167 DOI: 10.1159/000379753
Materials and Methods Animals Juvenile (30–35 days old, 50–70 g) and young adult (56–70 days old, 125–150 g) female Brown Norway rats (Charles River, Wilmington, Mass., USA) were used. The rats were housed under a 12-hour light-dark cycle. All rats were treated in accordance with the ARVO Statement for the Use of Animals in Vision Research.
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death has been studied in different experimental glaucoma models [1]. Diets high in fats and low in carbohydrates and proteins (so-called ketogenic diets, KDs) are one emerging non-pharmacological approach to treat neurodegenerative processes in disorders such as brain trauma, Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis [2, 3]. Although there are different variants of KDs, they all result in mimicking the biochemical changes that occur during starvation or extremely limited supply of carbohydrates with consequent changes in various metabolic and non-metabolic pathways related to energy metabolism, GABAergic inhibitory system, glutamatergic excitatory system, antioxidant mechanisms, programmed cell death, anti-inflammatory processes, and kynurenic acid production [2, 3]. Given that many of these mechanisms and related targets for neuroprotective strategies may also be relevant for glaucoma [1, 4] and are further supported by findings that calorie restriction facilitates the recovery of RGCs in acute intraocular pressure changes and ischaemia reperfusion models [5, 6], the present study investigated the effects of the KD on the survival of the RGCs in a rat model of excitotoxic damage to RGCs produced by Nmethyl D-aspartate (NMDA). This model is thought to reflect certain aspects of glaucoma; for example, both glaucomatous and ischaemic loss of RGCs in humans is discussed to be mediated, at least in part, by an overstimulation of NMDA receptors leading to intracellular calcium overload with consequent mitochondrial dysfunction and excessive formulation of free radicals [7]. Finally, given the reported age-dependent responses to KDs [8–11], the present study also explored the factor of age in the NMDA-induced damage to the RGCs in rats fed KDs.
Color version available online
a
b
c
d
e
f
g
h
Fig. 1. FluoroGold-labelled RGCs of rats 7 days after intravitreal PBS (a–d) or NMDA (e–h) injection: young rats fed SRD (a, e) or KD (b, f), adult rats fed SRD (c, g) or KD (d, h). Horizontal scale bar equals 50 μm.
Statistical Analysis Statistical analysis was performed using GraphPad Prism version 5.04 for Windows (GraphPad Software, San Diego, Calif., USA). Due to unequal variances for the compared treatment groups, the non-parametric two-tailed Mann-Whitney U test was used for statistical comparisons. Differences were considered significant when p < 0.05.
Results
RGC Density Seven days after intravitreal PBS injections, the densities of RGCs were comparable in juvenile and young adult rats fed either the SRD or KD for 21 consecutive days (table 1). Intravitreal NMDA injections resulted in a significant decrease in densities of RGCs in all groups tested regardless of the specific diet (table 1). However, the magnitude of NMDA-induced decrease in the density of RGCs was significantly lower (p = 0.005) in juvenile rats fed the KD when compared to those fed the SRD (table 1; fig. 1e, f). In contrast, NMDA-induced reduction in the density of RGCs was comparable (p > 0.05) in the young adult rats fed the SRD or KD (table 1; fig. 1g, h). 164
Ophthalmic Res 2015;53:162–167 DOI: 10.1159/000379753
Table 1. RGC density 7 days after intravitreal injections of PBS or
NMDA in juvenile and young adult rats fed SRD or KD for 21 days SRD
KD
Juvenile rats, PBS 2,822±73.9 (15) 2,606±79.3 (13) Juvenile rats, NMDA 307.4±25.7 (16) 416.3±22.6 (15)
Ophthalmic Res 2015;53:162–167 DOI: 10.1159/000379753
Ketogenic Diet Attenuates NMDA-Induced Damage to Rat’s Retinal Ganglion Cells in an Age-Dependent Manner Tomasz Zarnowski a Tomasz J. Choragiewicz a, d Frank Schuettauf d Eberhart Zrenner d Robert Rejdak a, c, d Maciej Gasior e Iwona Zarnowska b Sebastian Thaler d
a Tadeusz Krwawicz Chair of Ophthalmology and Eye Hospital and b Department of Pediatric Neurology, Medical University, Lublin, and c Department of Experimental Pharmacology, PAS Medical Research Center, Warsaw, Poland; d Eye Hospital and Institute for Ophthalmic Research, Centre for Ophthalmology, University of Tübingen, Tübingen, Germany; e Department of Pharmacology and Physiology, Drexel University College of Medicine, Philadelphia, Pa., USA
Abstract Objective: This study was conducted to investigate neuroprotective effects of a high fat/low carbohydrate and protein diet (ketogenic diet, KD) in a model of N-methyl D-aspartate (NMDA)-induced retinal ganglion cell (RGC) damage in juvenile and young adult rats. Methods: Juvenile (30–35 days old) and young adult (56–70 days old) female Brown Norway rats were fed the KD for 21 days; rats exposed to a standard rodent diet (SRD) served as controls. The main constituents of the KD used in the present study were approximately 80% fats, 8% proteins, and less than 1% carbohydrates. On day 14 of exposure to the KD (or the SRD in the control group), each rat received a single intravitreal injection of NMDA; RGCs were then retrogradely labelled by hydroxystilbamidine on day 19 and collected on day 21 to assess the degree of damage induced by NMDA. Blood biomarkers to confirm the expected metabolic response to the KD (i.e. ketosis and hypo-
© 2015 S. Karger AG, Basel 0030–3747/15/0533–0162$39.50/0 E-Mail [email protected] www.karger.com/ore
glycaemia) were also assessed. Results: Although both the juvenile and young adult rats developed comparable ketosis and hypoglycaemia when fed the KD, NMDA-induced loss in RGCs was significantly attenuated only in juvenile rats exposed to the KD in comparison with those fed the SRD; exposure to the KD had no protective effect in young adult rats. In summary, exposure to the KD had a neuroprotective effect in NMDA-induced RGC damage in juvenile rats, but not in young adult rats. Conclusion: These results support further exploration of metabolic interventions to treat optic neuropathies associated with neurodegeneration. © 2015 S. Karger AG, Basel
Introduction
Glaucoma is a leading cause of irreversible blindness and is the most common optic neuropathy caused by the accelerated death of retinal ganglion cells (RGCs). A role of neurodegenerative mechanisms related to excitotoxicity, mitochondrial dysfunction, misfolding of various proteins, oxidative stress, and inflammation in RGC Maciej Gasior, MD, PhD Department of Pharmacology and Physiology Drexel University College of Medicine Philadelphia, PA (USA) E-Mail NextPharma @ gmail.com
Downloaded by: Univ. of California San Diego 132.239.1.231 - 6/8/2015 2:19:50 PM
Key Words Ketogenic diet · Neuroprotection · Retinal ganglion cells · Rat
next 21 days. The KD (F3666 Ketogenic Diet for Rodents; Bio-Serv, Frenchtown, N.J., USA) consisted of 78.8% fats, 8.4% proteins, 5% cellulose, less than 5% water, and less than 1% carbohydrates, and was supplemented with necessary minerals and amino acids (detailed information available at supplier’s http://www.bio-serv. com/pdf/F3666.pdf). The KD of this specific composition has been commonly used in pre-clinical research as its composition closely mimics that of the classic KD used clinically in patients [2, 3]. To confirm the development of ketosis and hypoglycaemia in rats fed the KD, blood concentrations of β-hydroxybutyrate and glucose were measured in rats on day 14 of exposure to the KD and in the control rats for comparison using the Precision Xtra Blood Glucose and Ketone Monitoring System (Abbott, Alameda, Calif., USA) [12]. The blood sample was collected by making a small incision on the skin of the tail. Assessing the degree of ketosis and hypoglycaemia served as a qualitative assurance that the expected metabolic changes due to exposure to the KD in the present study were comparable to those reported elsewhere. Concentrations of glucose (mg/dl) and β-hydroxybutyrate (mmol/l) are presented as group means ± SEM. Intravitreal Injections Intraperitoneal injections of chloral hydrate (7%, 6 ml/kg body weight) were used for anaesthesia. On day 14, each rat received a single intravitreal injection of NMDA (Sigma-Aldrich, St. Louis, Mo., USA) consisting of 2 μl of a 10 mmol/l NMDA solution in PBS using heat-pulled glass capillaries connected to a microsyringe (Drummond Scientific Co., Broomall, Pa., USA) under direct observation through the microscope. The NMDA dose was chosen in order to gain a reduction of healthy RGCs of about 90% in RGC counts of otherwise untreated animals, comparable to earlier studies [13]. This allowed studying potential neuroprotective effects of the KD under the worst scenario of nearly complete RCG damage. Rats in which lens damage occurred during this procedure were excluded from the study and were not used thereafter. In each case, the contralateral eye served as a control eye and was injected with PBS.
Diets Rats were fed ad libitum with a standard rodent diet (SRD, AIN-93G Basic Diet 2222 for Small Rodents; Kliba-Nafag, Kaiseraugst, Switzerland) before being divided into two groups. One group continued to be fed the SRD (control group) until the end of the experiment. The other group started to be fed the KD for the
Retrograde Labelling and Quantification of RGCs Five days after intraocular injection (day 19), the rats were anaesthetized, and a total of 7 μl of a fluorescing dye (hydroxystilbamidine methanesulfonate; Molecular Probes, Eugene, Oreg., USA) was applied to each superior colliculus with three stereotaxic-guided injections of 2.33 μl each at a depth of 3.5 mm (below brain surface). The injection site was a 1.5 × 2.5-mm rectangular window located between lambda and the bregma suture of the skull. On day 21 rats were sacrificed with CO2, the corneas of the treated eyes were marked for orientation in the form of retinal quadrants. The eyes were then enucleated, and the retinas were dissected, flat-mounted, and fixed in 2% paraformaldehyde. Visualization was performed immediately under a fluorescent microscope. Counting was carried out in 4 distinct areas (62,500 μm2 each) in the middle periphery of the retina corresponding to 2.0 mm distance from the optic nerve head at the 45, 135, 225, and 315° meridian, as described in detail previously [13]. Only one defined distance to the optic nerve was chosen in order to reduce animal numbers and increase sensitivity, due to the fact that RGC numbers are not uniformly distributed from the central to the peripheral regions of the rat retina [14]. Images were obtained using a digital imaging system connected to the microscope (ImagePro;
Ketogenic Diet and Retinal Ganglion Cell Damage in Rats
Ophthalmic Res 2015;53:162–167 DOI: 10.1159/000379753
Materials and Methods Animals Juvenile (30–35 days old, 50–70 g) and young adult (56–70 days old, 125–150 g) female Brown Norway rats (Charles River, Wilmington, Mass., USA) were used. The rats were housed under a 12-hour light-dark cycle. All rats were treated in accordance with the ARVO Statement for the Use of Animals in Vision Research.
163
Downloaded by: Univ. of California San Diego 132.239.1.231 - 6/8/2015 2:19:50 PM
death has been studied in different experimental glaucoma models [1]. Diets high in fats and low in carbohydrates and proteins (so-called ketogenic diets, KDs) are one emerging non-pharmacological approach to treat neurodegenerative processes in disorders such as brain trauma, Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis [2, 3]. Although there are different variants of KDs, they all result in mimicking the biochemical changes that occur during starvation or extremely limited supply of carbohydrates with consequent changes in various metabolic and non-metabolic pathways related to energy metabolism, GABAergic inhibitory system, glutamatergic excitatory system, antioxidant mechanisms, programmed cell death, anti-inflammatory processes, and kynurenic acid production [2, 3]. Given that many of these mechanisms and related targets for neuroprotective strategies may also be relevant for glaucoma [1, 4] and are further supported by findings that calorie restriction facilitates the recovery of RGCs in acute intraocular pressure changes and ischaemia reperfusion models [5, 6], the present study investigated the effects of the KD on the survival of the RGCs in a rat model of excitotoxic damage to RGCs produced by Nmethyl D-aspartate (NMDA). This model is thought to reflect certain aspects of glaucoma; for example, both glaucomatous and ischaemic loss of RGCs in humans is discussed to be mediated, at least in part, by an overstimulation of NMDA receptors leading to intracellular calcium overload with consequent mitochondrial dysfunction and excessive formulation of free radicals [7]. Finally, given the reported age-dependent responses to KDs [8–11], the present study also explored the factor of age in the NMDA-induced damage to the RGCs in rats fed KDs.
Color version available online
a
b
c
d
e
f
g
h
Fig. 1. FluoroGold-labelled RGCs of rats 7 days after intravitreal PBS (a–d) or NMDA (e–h) injection: young rats fed SRD (a, e) or KD (b, f), adult rats fed SRD (c, g) or KD (d, h). Horizontal scale bar equals 50 μm.
Statistical Analysis Statistical analysis was performed using GraphPad Prism version 5.04 for Windows (GraphPad Software, San Diego, Calif., USA). Due to unequal variances for the compared treatment groups, the non-parametric two-tailed Mann-Whitney U test was used for statistical comparisons. Differences were considered significant when p < 0.05.
Results
RGC Density Seven days after intravitreal PBS injections, the densities of RGCs were comparable in juvenile and young adult rats fed either the SRD or KD for 21 consecutive days (table 1). Intravitreal NMDA injections resulted in a significant decrease in densities of RGCs in all groups tested regardless of the specific diet (table 1). However, the magnitude of NMDA-induced decrease in the density of RGCs was significantly lower (p = 0.005) in juvenile rats fed the KD when compared to those fed the SRD (table 1; fig. 1e, f). In contrast, NMDA-induced reduction in the density of RGCs was comparable (p > 0.05) in the young adult rats fed the SRD or KD (table 1; fig. 1g, h). 164
Ophthalmic Res 2015;53:162–167 DOI: 10.1159/000379753
Table 1. RGC density 7 days after intravitreal injections of PBS or
NMDA in juvenile and young adult rats fed SRD or KD for 21 days SRD
KD
Juvenile rats, PBS 2,822±73.9 (15) 2,606±79.3 (13) Juvenile rats, NMDA 307.4±25.7 (16) 416.3±22.6 (15)
