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Corresponding author at: Department of Medicine and Surgery, University of Salerno, via S. Allende snc, 84081 Baronissi (SA), Italy. Tel.: +39 089965083.
Department of Molecular Pharmacology and Physiology, Hyperbaric Biomedical Research Laboratory, Morsani College of Medicine, University of South Florida, 12901 Bruce B. Downs Blvd., MDC 8 Tampa, FL, United StatesExternal Pharmacy of Fatebenefratelli Hospital, Viale Principe di Napoli 14/A, Benevento, Italy
Department of Molecular Pharmacology and Physiology, Hyperbaric Biomedical Research Laboratory, Morsani College of Medicine, University of South Florida, 12901 Bruce B. Downs Blvd., MDC 8 Tampa, FL, United States
Department Medicine and Surgery, University of Salerno, via Allende snc, Baronissi (SA), ItalyUO Child and Adolescent Neuropsychiatry, Medical School, University of Salerno, Largo Città di Ippocrate snc, Salerno, Italy
The anti-seizure efficacy of a prolonged administration of a synthetic ketone has been evaluated.
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The prolonged administration of 1,3-butanediol acetoacetate diester by gavage produces calorie restriction in rats.
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Gavage treatments in rats produce calorie restriction and anti-seizure effects similar to that of a ketogenic diet.
Abstract
Purpose
Previous studies showed that a single oral administration of a synthetic ketone ester (1,3-butanediol acetoacetate diester, BD-AcAc2) could elevate blood ketones with promising acute anti-epileptic effects. The aim of the present work was to evaluate the tolerability of a prolonged administration of BD-AcAc2 and the anti-epileptic efficacy of such treatment.
Methods
The threshold for seizure induction with progressive intravenous infusion of pentylenetrazole (PTZ) was evaluated in anesthetized Wistar rats after a ten-day oral administration of BD-AcAc2 (gavage). The effects of this treatment were compared to those of: (1) a ten-day water gavage administration, (2) a ten-day ketogenic diet, (3) a standard rodent chow diet.
Results
Compared to the standard diet, all other treatments produced a calorie restriction and an elevation of the seizure threshold.
Conclusion
These results indicate that supplementation with an oral synthetic ketone can have anti-seizure effects, but the formulation has to be further ameliorated to be more palatable; further studies are also needed to better understand the role played by ketone bodies alone in vivo, without any calorie restriction.
]. One hypothesis has been that ketone bodies, typically increased during a KD, can have an anti-epileptic effect. Accordingly, in previous studies it has been shown that a single oral administration of a synthetic ketone ester (1,3-butanediol acetoacetate diester, BD-AcAc2) had promising acute anti-seizure effects, as assessed by a hyperbaric-induced convulsion model [
], thus it is questioned how much the anti-epileptic effect of KD can be ascribed to ketone bodies alone.
The aim of the present work was to evaluate the tolerability and the anti-seizure efficacy of a prolonged administration of BD-AcAc2 in the PTZ-model, comparing this effect with that of a calorie-restricted KD.
2. Materials and methods
2.1 Animals
Twenty male Wistar rats (Harlan Laboratories, Udine, Italy), weighting 150 ± 15 g (mean ± S.D.), were randomly divided into four groups. Rats were housed in standard temperature and humidity conditions with a 12 h light–dark cycle (lights on from 07:00 a.m. to 07:00 p.m.). Animals belonging to groups 1, 2 and 3 were given free access to standard food and tap water. Animals belonging to group 4 were, instead, given an anti-epileptic calorie-restricted KD [
]. All procedures fulfilled the requirements of the “Guide for the Care and Use of Laboratory Animals‘’ released by the National Research Council and implemented by EU and local rules. Animals were kept under general anesthesia with urethane during all surgical procedures and seizure induction; the research was approved by the local ethical committee of the University of Salerno and by the Ministry of Health of the Italian Government.
2.2 Pre-treatment
Animals of group 1 and group 2 were fed with a standard diet and were given by gavage 1 ml of water (group 1) or BD-AcAc2 (group 2) two times per day (at 09:30 a.m. and at 04:00 p.m.) for ten days. This dose was chosen because previous studies demonstrated that 1 ml of BD-AcAc2 has acute anti-seizure effects [
]; the dose was administered twice daily because the elevation in blood ketones does not last more than 8 h (unpublished data). Details on BD-AcAc2 synthesis are reported elsewhere [
]. Rats of group 3 were not given any gavage and received a standard diet. Rats of group 4 were not given any gavage but were fed with a calorie-restricted KD that has a well documented anti-seizure effect [
]; briefly, each day they were given approximately 90% of the calculated daily calorie requirement by a food mix composed of more than 78% fat (Bio-Serv cF3666, Frenchtown, NJ, USA).
Body weights were recorded every day. Blood glucose (GlucoMen LX plus, Menarini Diagnostics, Firenze, Italy) and blood ketones in the form of β-hydroxybutyrate (βHB; Freestyle Optium, Abbott, Rome, Italy) were recorded on day 0 (start of treatment), and on days 2, 5 and 7; blood drops were obtained by tail prick at 09:00 a.m. (before the morning gavage).
Possible neurotoxic effects were evaluated in all rats on days 0, 3 and 8 through five behavioral tests, as described by Swinyard et al. [
]: positional sense test, righting-reflex test, gait-and-stance test, muscle tone, equilibrium. For each test a score of 0 (failed), 1 (positive) or 2 (quick response) was assigned, thus the total score (i.e. the sum of the scores from the 5 tests) could range from 0 to 10.
2.3 Surgery
On the day of the experiment, weight, blood glucose and blood ketones were again recorded for each rat and a gavage of 1 ml of water (group 1) or BD-AcAc2 (group 2) was administered. Thirty minutes after the gavage, rats were anesthetized with 1.5 g/kg b.w. of urethane (Sigma–Aldrich, Milano, Italy); rats from group 3 (standard diet) and group 4 (KD) were anesthetized without gavage. From this time until the end of the experiment, body temperature was maintained at 36.5 ± 0.5 °C with a thermostated heating pad. The femoral vein was then accessed by inguinal incision and insertion of a polyethylene catheter (outer diameter 0.6 mm) to a depth of 6 cm. Placement of electrodes to record cortical-electroencephalogram (cEEG) was achieved by percutaneous incision of the scalp and osteotomy of the calvarium. Two holes (1.4 mm in diameter) were drilled on the skull at the following coordinates: first hole at 2 mm lateral from midline and 2 mm anterior from bregma; second hole at 2 mm lateral from midline and 2 mm posterior from bregma. Two stainless steel screws (1.6 mm in diameter; Novara Metalli, Novara, Italy) were placed into these holes at a depth of 2 mm from the skull surface and served as recording electrodes. A safety pin, the reference electrode, was placed subcutaneously in the posterior of the neck.
2.4 cEEG recording
The recording and reference electrodes were connected to a custom made digital amplifier [
], which provided a gain of 5000 v/v, a band pass filter from 1.6 Hz to 100 Hz and an analog-to-digital conversion with a resolution of 12 bits and a sampling frequency of 1000 samples/s. The amplifier sent the digital readings to a PC through a standard USB port; a custom software written with LabView (National Instruments, Austin, Texas, USA) provided real time visualization and recording of the cEEG for subsequent analysis and allowed manual marking of the events of interest (e.g. perfusion start and perfusion stop).
At 1 h:00 min after the anesthesia, cEEG recording was started. At 1 h:30 min after the anesthesia, when BD-AcAc2 gavage has the maximum effect on blood βHB levels [
], the femoral catheter was connected to a syringe-pump prefilled with 100 mg/ml of PTZ (Sigma–Aldrich, Milano, Italy), and PTZ infusion was started at a rate of 0.25 ml/min. When a sustained high frequency and high amplitude seizure discharge was evident (greater than 0.5 mV from the zero level of the signal and lasting more than 5 s), the infusion was interrupted and recording was maintained for another 30 min. The protocol time sequence is summarized in Fig. 1A.
Fig. 1(A) Time sequence of the experimental protocol. Infusion was stopped when a sustained seizure discharge was present. (B) Examples of cEEG recordings from a no-gavage/standard diet-fed rat (control) and from a KD-fed treated rat. The start of the PTZ injection is marked with the longer arrows; PTZ was injected with a constant rate of 25 mg/min. The PTZ-threshold was evaluated as the time between PTZ infusion start (longer arrows) and the time of the first spike indicating a seizure-like firing (short arrows); this spike was defined as having an amplitude greater than 0.5 mV (in both positive and negative phases) and with an inter-peak latency shorter than 1 s. In these particular examples, the PTZ dose injected until the above defined PTZ-threshold were 40 mg/kg b.w. for the control rat and 112 mg/kg b.w. for the KD-fed rat.
For each cEEG recording, the onset-time of the seizure discharge was measured, defined as the time from the infusion start to the first of the above-threshold peaks; above-threshold peaks were defined as having an amplitude greater than 0.5 mV, with inter-peak latency time lower than 1 s (Fig. 1B). For each rat, the PTZ-threshold for seizure induction was defined as the injected dose of PTZ at the onset-time of the seizure discharge (mg/kg b.w.).
2.5 Histological controls
At the end of the seizure recording procedures, rats were sacrificed by anesthetic overdose. Liver, kidney and stomach were immediately removed and stored in 4% formaldehyde/phosphate buffer solution. Tissues were processed by routine histological procedures and stained with hematoxilin/eosin.
2.6 Statistical analysis
Data are expressed as mean ± S.E. Statistically significant differences were evaluated by the analysis of variance (ANOVA) for repeated measures; post hoc test – multiple comparisons were performed with the Tuke's test.
3. Results
3.1 Body weight
All groups differed from each other when compared for body weight gain (Fig. 2); compared to the no-gavage/standard diet-fed group (group 3), all other groups showed a strong impairment in body weight gain. The grading for the weight gain of the treatments was BD-AcAc2-gavage < water gavage < KD << standard diet. The ANOVA for repeated measures demonstrated a significant effect for the treatment (F3,144 = 2011, P < 0.01), for the time (F8,144 = 10, P < 0.01) and for the treatment × time interaction (F24,144 = 292, P < 0.02). Multiple comparisons showed that all groups differed from each other from day 3 to day 9, except for the comparison water-gavage group – KD group which was significant from day 6 to day 9 (P < 0.05).
Fig. 2Mean weight trends of the experimental groups. Values are expressed as the percentage of weight at day 0. All groups differed from each other from day 3 to day 9, except for the comparison water-gavage group – KD group, which was significant from day 6 to day 9 (P < 0.05).
All blood glucose values were within the normal range (100 ± 25 mg/100 ml) and there were no significant differences between groups.
3.3 Blood βHB
As expected, the KD-group showed higher blood βHB values than those observed in the controls (Fig. 3); all other groups showed normal low values for blood βHB with no significant differences between them. The ANOVA for repeated measures showed a significant effect for the treatment (F3,64 = 37, P < 0.01) and for the treatment × time interaction (F9,64 = 4.2, P < 0.01). The post hoc test demonstrated a significant difference between the KD-treated group and all other groups (P < 0.05).
Fig. 3Mean blood βHB level from all experimental groups. Values were taken at morning, before any gavage, thus showing the minimal levels produced by the treatments. Only the KD group had significantly high blood ketone levels. Asterisks indicate statistical significant difference compared to other groups at that time point (P < 0.05).
No rat failed any behavioral test at any time point. No statistically significant difference was seen for the total score between the experimental groups.
3.5 Seizure PTZ-threshold
Compared to the no-gavage/standard diet group, all other groups showed a higher PTZ-threshold (Fig. 4). The ANOVA showed a significant difference between groups (F3,16 = 5.02, P < 0.02). The post hoc test demonstrated a significant difference between the no-gavage/standard diet group and all other groups; there were no significant differences between the KD group, the BD-AcAc2 group and the water-gavage group (Fig. 4).
Fig. 4Mean seizure PTZ-threshold obtained from each experimental group. All treatments resulted in a higher threshold compared to the no-gavage/standard diet-fed group (control). The asterisk indicates statistical significant difference compared to other groups (P < 0.05).
Morphological findings were mostly normal for all the organs. Some renal peri-tubular blood extravasations were observed in one rat from the BD-AcAc2-treated group and in two rats from the water-treated group. A tubular necrosis and a partial glomerular sclerosis were also observed in another rat from the BD-AcAc2-treated group (Fig. 5).
Fig. 5Kidney histological control. (A) A blood extravasation (arrow) in the medulla detected in one BD-AcAc2-treated rat. The same was observed in two water treated rats. (B) A glomerular sclerosis (long arrow) and tubular necrosis (point arrow) were seen in a BD-AcAc2-treated rat.
The results from the present experiments demonstrated that many different treatments causing a calorie restriction can also produce an anti-seizure effect when evaluated with the anesthetized PTZ-model. In fact, even a water gavage treatment, which resulted in growth retardation (Fig. 2) probably due to stress from manipulation and decreased food intake, resulted in an increased seizure PTZ-threshold compared to control animals to an extent comparable to that obtained with a KD (Fig. 4). This result does not support a major role for ketone bodies in an anti-seizure effect, because three very different treatments produced a comparable anti-seizure affect (KD, water gavage, BD-AcAc2 gavage), but only the KD treatment resulted in a significant increase in blood ketones. Nevertheless, an effect of ketone bodies cannot be excluded, because even in humans just a restricted diet (or fasting) with water contributes to ketosis and is frequently used before initiating a KD. Moreover, at present we failed to obtain an increase in blood ketones without a significant calorie restriction; further attempts should be made to obtain this condition, thus evaluating the anti-seizure efficacy of ketone bodies alone in vivo.
To better test BD-AcAc2 as an anti-seizure medication, future experiments should be carried out to verify if it is possible to obtain an anti-seizure effect without any calorie restriction by trying to administer this compound in a different way, such as mixed with the food pellets or as a different, water-soluble pharmaceutical preparation of the active ingredient (ketone mineral salt). BD-AcAc2 did not show toxic effects on liver, kidney, stomach, or neurological functions. Minor abnormal histological findings were reported in two BD-AcAc2 -treated rats and in two water-treated rats, thus these effects were unlikely caused by BD-AcAc2; further studies on a larger number of observations are needed to eventually prove a correlation between BD-AcAc2 and kidney pathologies. However, the administration of BD-AcAc2 produced a partial but significant anorectic effect; thus, the general tolerability of this pharmaceutical preparation, at least in terms of palatability, is unlikely better than that of KD. A smaller dose could be more tolerable, but further studies should verify if an anti-seizure effect persists, given that even the dose used in the present experiment did not cause a 24 h elevation in blood ketones (Fig. 3). This is another reason for trying different, more palatable pharmaceutical preparations, like a water soluble ketone salt.
The anti-seizure effect seen in all calorie restriction conditions in these experiments agrees with previous findings in young rats [
]. Actually, there were many historical reports noting an effect for calorie-restriction, and in particular for fasting, since the Hippocratic collection; in the modern era, this notion was first drawn to the attention of the scientific community by H. Rawle Geyelin [
]. In present days, calorie-restriction is still acknowledged for having many positive effects on longevity, diabetes, hypertension, cardiovascular disease, and cancer [
]; nevertheless, the mechanism by which calorie restriction produces anti-seizure effects has still to be clarified.
In conclusion, from the present results three objectives emerge for future research: (1) to ameliorate animal models that mimic modern clinical dietary treatments, i.e. calorie restriction without growth impairment; (2) to find in vivo specific anti-seizure effects of ketone bodies alone; (3) to formulate more tolerable synthetic ketone bodies supplementations.
Conflict of interest
All the authors state that there are no conflict of interest.
Acknowledgments
The present experiments were supported by the FARB fund (Prof. Coppola) and the starting fund (Prof. Viggiano) by the University of Salerno. Thanks to: Dr. Valentina Iovane and Mr. Luigi De Lucia for animal care support; the M.D. students Barbara Buonomo and Marianna Perrotta for helpful assistance with daily activities and Ms. Madison Stoddard for helpful suggestions in reviewing the manuscript.
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