Effects of a Ketogenic Diet on the Evaluation of Biochemical and Clinical Parameters of Duchenne Muscular Dystrophy: a Preclinical Study

doi:10.21203/rs.3.rs-1965435/v1

Download PDF

Research Article

Effects of a Ketogenic Diet on the Evaluation of Biochemical and Clinical Parameters of Duchenne Muscular Dystrophy: a Preclinical Study

https://doi.org/10.21203/rs.3.rs-1965435/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Duchenne muscular dystrophy (DMD) is one of the X-linked recessive progressive muscular dystrophies, characterized by progressive degeneration of skeletal muscle and multisystemic involvement, including the central nervous system (CNS). To investigate the effects of DCet on biochemical and clinical parameters in a mouse model with DMD. Methods: Young adult mice (50 days old) were fed DCet for 14 days; control groups received a standard diet for the same period. On the 14th day of exposure to diets, each mouse was submitted to memory and behavior evaluation tests. After the behavioral tests, the animals were sacrificed for biochemical evaluation of oxidative stress and levels of inflammatory biomarkers. Body weight, feed intake and BDNF levels were also evaluated. Results: mdx+DCet mice had less mass (0.2g ± 2.49) and presented memory retention (p <0.05) in relation to the control group. There was reduction of oxidative damage in muscle tissue and CNS, and significant decrease in cytokine levels (p <0.05). This protocol increased the level of hippocampal BDNF and mitochondrial respiratory complex activity in both muscle tissue and CNS, although it only decreased creatine kinase activity in the striatum.

Duchenne muscular dystrophy

Mdx

Central nervous system

Muscle

Duchenne muscular dystrophy (DMD) is one of the X-chromosome-linked progressive muscular dystrophy (PMD), is recessive and is expressed symptomatically in male family members. It occurs in 1 in 3,600 live male births and is the most common fatal genetic disease in children [1, 2]. It is characterized by progressive and debilitating muscle weakness caused by the absence of the dystrophin protein. This absence increases the muscle fiber membrane fragility during contraction, resulting in repeated cycles of necrosis and regeneration and the final replacement of the injured tissue by fibrous and adipose connective tissue [3].

While significant progress has been made in scientific advancement on DMD, there is still no cure for the disease. Corticosteroid therapy is the standard drug treatment currently used, and corticoids are the only pharmacological agents with documented benefits, despite being associated with several adverse effects, including nutritional ones. In this case, non-drug therapies aimed at minimizing the disease progression have acquired significant importance [4, 5].

Since 1921, the ketogenic diet – with low carbohydrate content, 10% protein and high fat content – has been successfully used for the treatment of refractory childhood epilepsy to foster antiepileptic [6, 7] and neuroprotective [8, 9, 10] effects. Furthermore, this type of diet was able to increase mice mitochondrial antioxidant status by regulating the reduced glutathione (GSH) biosynthesis [11]; it slowed the progression of mitochondrial myopathy in animal models [12]; it inhibited oxidative damage and showed improvement in mitochondrial bioenergetics in brain tissue of animal models and it protected from neurodegenerative sequelae [11, 13]. In addition, investigators showed that there was modulation of the inflammatory process in rats injected with lipopolysaccharides (LPS - E. coli) with reduced expression of interleukin 1 beta (IL-1β) in brain tissue [14]. In children with autism [15], the use of this therapy evidenced beneficial effects.

Thus, for a systemic pathology such as DMD, nutrition is not expected to be the only treatment approach, but more accurate dietary recommendations along with other therapies could help manage the disease and slow damage progression. With a severe dystrophin deficiency, subtle dietary changes may not overcome any significant portion of cellular insult, but it is possible that considerable changes in dietary composition could alter the severity of the disease. Thus, the aim of this study was to verify the impact of the ketogenic diet on the DMD pathophysiological process.

ANIMAL MODEL

Adult mdx mice and C57bl/6 (wt) mice were used in their adult phase (50 days of life). The C57bl/6 wild type animals were supplied by the Federal University of Santa Catarina (UFSC); and, the mdx animals came from the University of São Paulo (USP). The study was conducted at Lanex and at the Laboratory of Biochemistry and Molecular Biology of Unisul, Palhoça, Santa Catarina, Brazil. Approval for conducting this study was obtained from the Animal Use Ethics Committee (CEUA) of Unisul under the number 16.051.4.01IV.

The animals were kept in polypropylene boxes of adequate dimensions, with wood shavings, in an acclimatized room (22°C ± 2°C), with a 12-hour timer-controlled light/dark cycle; they were given water and a standard ad libitum diet throughout the experiment period.

The number of animals per group was set at n=8. The formula used for the calculation was the equation n /group=2[(Zα/2 +Zβ) x d/∆]2., for the comparison of two means, considering a test power of 80% , a 5% significance level, a 12.5 % standard deviation based on the records of previous studies and the value of the difference to be detected equal to 18%.

Upon completing 50 days of life, the animals were divided into four experimental groups: wt+DC, wt+DCet, mdx+DC, mdx+DCet and fasted for 8 hours before the beginning of the procedures. After this period, they were exposed to two types of diet for 14 days [8,14,16,17]: ketogenic diet (DCet) or control diet (CD).

Regarding the diets offered, two diets with different nutritional compositions were used: pelleted CD containing 22% proteins, 10% total fat and 68% carbohydrates (Nuvilab® CR1 Nuvital Nutrientes S/A, Brazil); DCet, not pelleted, with 10% protein, 90% total fat and 0.1% carbohydrates (PragSoluções®, Brazil) (Table 1).

Table 1 - Nutritional composition of the diets under study, per 100g portion.

Estimated minimum¹		Control diet²		Dketogenic diet³
Calories (kcal)	360	398		676
	%	g	%	g	%
Proteín	14	22	22,1	17,6	10,4
Lipids	10	4,5	10,2	67,2	89,5
Carbohydrates	76	67,3	67,7	0,2	0,1

(1)AIN-93M107. (2)Control diet = standard ration. (3)Ketogenic diet = test feed (PragSoluções®).

On the 14^th and 15^th day, the animals underwent behavioral tests, followed by painless assisted death (PAD) on the 15^th day.

Measuring body mass feed consumption

Body mass was measured twice a week during the diet therapy period, at the same time of day by trained investigators using a digital scale. The mass gain control was performed by simple variation (final – initial) and by the rate of mass gain following the formula: Δ% = [(final mass - initial mass/initial mass)x100].

The amount of feed offered was estimated according to the number of animals in each box and considering the average amount of feed consumed per animal per day of approximately 6g (+ 10% waste)[18]. The animals from the same group were randomly distributed, separated by gender, at 1< n ≤5, as social isolation modifies the biochemical profile and consumption pattern of experimental animal models [19].

Memory and learning

Inhibitory Avoidance

The assessment of avoidance memory consisted of two steps: training and testing, performed at an interval of 24 hours to assess long-term aversive memory. The assessment was carried out by the same investigators using a specific equipment for such assessment: the passive avoidance box. In the training session, the animals were placed on the platform and the time that the animal took to descend with all four paws from the platform was measured. This period of time is called latency. Immediately after coming down from the platform (with all four paws), the animal received a shock of 0.2 mA for 2 seconds. In the test session, the animal was again placed on the platform and the time taken to descend (latency) was measured, but without shock administration. Latency is a classic memory retention parameter [20].

Open-field habituation

The assessment of open field habituation took place in two stages: training and testing. It was performed at an interval of 24 hours, by two trained researchers using specific equipment and observation techniques. In the training session, one animal at a time was carefully placed in the left posterior quadrant by one of the investigators, from which the exploratory behavior of the setting was observed freely for 5 minutes. In the test session, the animals were placed individually in the exploratory box again for the same assessment. The number of crossings across the quadrants (crossing: motor activity) and the number of rearings (exploratory activity, in which the animal stands on two paws) were evaluated in both sessions[20].

Collection of biological material

Immediately after PAD by anesthetic deepening (intraperitoneal application), samples of structures were removed: from the brain tissue which are associated with learning and memory and where they present greater expression of dystrophin [21].(prefrontal cortex, cerebellum, hippocampus, striatum and total cortex) by surgical dissection; blood, by cardiac puncture to assess ketonemia; and, of muscle tissue (gastrocnemius) with the objective of control as it is a neuromuscular pathology also by surgical dissection. Samples were kept at negative 80ºC for subsequent biochemical analyses.

Biochemical determinations

Cytokines (TNF-α, IL-1β IL-6 and IL-10) concentrations were analyzed by the ELISA method using specific standard kits, following the manufacturer's recommendations and using replicate dosing.

Thus, in short, 96-well plates were sensitized with 100 μl of captured antibody diluted in a coating buffer and incubated under refrigeration overnight for antibody adhesion. After that period, the wells were washed three times with 250 μl buffer. Subsequently, the plate was blocked with 100 µl of blocking solution (1% bovine serum albumin (BSA) in sterile phosphate-buffered saline (PBS), pH 7.2) to avoid unspecific binding and was incubated for 1 hour at room temperature. . At the end of this period, the wells were washed again, as described above.

After blocking, 100 µl of the samples diluted 1:3 in dilution reagent (1% BSA in sterile PBS, pH 7.2) were added and placed again in a moist chamber and incubated overnight at room temperature. The samples were plated in duplicate and 100 µl of the dilution solution was placed in a well for blank characterization. After this period the plates were washed as previously described.

After washing, 100 µl of detection antibody previously diluted in 1:3 reagent (1% BSA in sterile PBS, pH 7.2) was added to each well. The plate was incubated at room temperature for 1 hour. The wells were washed again as described above. Subsequently, 100µl of streptoavidin-HRP was added in each well. The plate was incubated in the dark at room temperature for 30 minutes. Afterwards, the wells were washed once more. Subsequently, 100 µl of the substrate solution (color reagent A-H₂O₂ and color reagent B-OPD 1:1) were added in each well, and incubated for 30 minutes protected from light at room temperature. The reaction was stopped with 50 µl of H₂SO₄(2N) per well. Absorbance was measured by spectrophotometry at a wavelength of 490nm.

Myeloperoxidase assay

The presence of tissues neutrophils was indirectly measured through the quantification of the MPO enzyme activity. Hence, the selected tissues were first homogenized in a PBS-HTAB buffer solution (100 µL for each 50 mg of tissue). The samples were centrifuged for 15 minutes at 4000 rpm at 4°C. The supernatant was collected and centrifuged again at 4000 rpm for another 15 min. A 50 µL homogenizing solution (PBS, 5 µM diaminotetraacetic acid (EDTA), 0.5% HTAB) was pipette as well as 50 µL of the supernatant and 50 µL of 0.68 mg/mL o-dianisidine (1 tablet of o-dianisidine 10 mg, 14.7 mL milli-q water) in duplicate in the 96-well plate. The plate was incubated at 37°C in the reader for 15 min and after this period 50 µL of H₂O₂ solution was added. The incubation process was repeated for another 10 min at 37ºC and then read in a spectrophotometer at 460 nm. For the blank, only 200 µL of substrate buffer were used. To determine the MPO value, a standard curve was drawn, using predetermined concentrations, in the equation: y = 1.2358x+ 0.252 where y is the concentration of MPO in ng/mL and x is the value found from the final reaction derived from the cell suspension.

Measurement of substances reactive to thiobarbituric acid (TBARS)

The lipid peroxidation rate was evaluated by measuring the substances reactive to TBARS. For this analysis, the protocol described by Draper and Hadley (1990) [22] was followed. This method is based on the reaction of two molecules of thiobarbituric acid with one of malondialdehyde (MDA), producing a pink color complex that can be quantified in a spectrophotometer. This reaction occurs at acidic pH and at temperatures between 90 and 100ºC. An aliquot of 100 µL of brain and muscle tissue was homogenized with 200 to 1000 µL of a solution of trichloroacetic acid (10%) - thiobarbituric acid (0.067%), heated for 15 minutes in boiling water and cooled in ice water, centrifuged for 10 minutes. The supernatant was used to quantify the TBARS using the spectrophotometer at a wavelength of 535nm. The results obtained were compared with a MDA standard curve. The TBARS concentration was determined using 1.56x10⁵xM^-1cm^-1 as the molar extinction coefficient of MDA. Values were expressed in ng of TBARS/mL of tissue [22].

Measurement of oxidative damage in proteins

Oxidative damage to tissue proteins was determined, spectrophotometrically, by measurement of the carbonyl groups according to the standard protocol described by Levine et al. (1990) [23]. The samples obtained were precipitated by adding 20% trichloroacetic acid and the proteins dissolved with DNPH. The absorbance of the carbonyl groups content was spectrophotometrically measured at 280 nm. The concentration of carbonylated proteins was expressed in nmol/milligram of protein [23].

Total content of free thiols

In order to analyze the levels of free thiols, the samples were precipitated with trichloroacetic acid (10%), centrifuged and the supernatant was added to 5.5' Dithiobis-2-nitrobenzoic-1,7 (DTNB) and tris-HCl buffer (30 mM , containing 3 mM EDTA, pH 8.9), generating a yellow-colored derivative and read in the spectrophotometer at 412nm. This technique aims to evaluate the non-oxidized sulfhydryl (SH) group. The results were expressed in free thiols nmol/mg of protein [24].

Activity of mitochondrial respiratory chain complexes

To determine the activity of the enzymatic complexes of the respiratory chain, the brain structure was homogenized (1:10, w/v) in buffer pH 7.4. The homogenate was centrifuged at 800xg for 10 minutes and the supernatant stored at 80°C negative to determine the enzymatic activity.

Complex I activity was evaluated by the method described by Cassina and Radi (1996) [25] by the NADH-dependent rate of ferricyanide reduction at 420 nm. CK activity was determined according to Hughes methodology (1962)[26] and measured at 550 nm. The activity of the complex was expressed in nmol/min.g per protein, while the CK activity was expressed in mg/protein.

Quantification of BDNF levels

BDNF levels were measured by an anti-BDNF ELISA sandwich kit according to the manufacturer's instructions. In short, for this analysis, the samples were homogenized in phosphate buffer solution with 1mM of phenylmethylsulfonyl fluoride and 1mM of glycol diaminetetraacetic acid (EGTA). The 96-well of the microtiter plates (flat bottom) were coated for 24 hours with samples diluted 1:2 in sample diluent and using the standard curve ranging from 7.8 to 500pg/ml BDNF. The wells were then washed four times with sample diluents, and a rabbit anti-BDNF monoclonal antibody diluted 1:1000 in sample diluent was added to each well and incubated for 3 hours at room temperature.

PROCESSING AND DATA ANALYSIS

For statistical analysis, the Prism program, GraphPad Software was used. The Shapiro-Wilk test was previously applied to check the data normality. Data from the open field habituation test and biochemical assessments were considered parametric and expressed as mean and standard deviation of the mean. Statistical comparisons between groups were performed by one-way analysis of variance (ANOVA) followed by the Tukey's test. Statistical comparisons between two factors were analyzed by the t test for paired samples. Data referring to the inhibitory avoidance test were considered non-parametric and were expressed as median and interquartile range; statistical comparisons between two factors were performed using the Mann-Whitney test. For all the analyses, differences were considered statistically significant when p<0.05.

BODY MASS AND FEED CONSUMPTION

The initial and final body mass mean are described in Table 2, showing the weight gain during the period of diet therapy in all groups.

Table 2

Average body mass and weight gain per group
Groups	Initial (g)	Final (g)	Body mass gain (final - initial) (g)	Weight gain (%)
wt + DC	22,4 ± 2,48^A	23,5 ± 2,54^B	1,4 ± 2,40^ac	4
wt + DCet	17,8 ± 4,01^A	19,7 ± 3,43^B	1,9 ± 2,54^ab	20
mdx + DC	14,9 ± 2,15^A	18,8 ± 1,85^B	3,8 ± 3,00^b	36
mdx + DCet	18,8 ± 3,53^A	19,0 ± 3,00^A	0,2 ± 3,49^c	4

Different lowercase letters in the same column indicate significant differences between groups. Different capital letters on the same line indicate significant differences between final and initial body mass in the same group. Results presented as mean ± standard deviation (p < 0.05).*ANOVA, Tukey post-test.

At the end of 14 days experimental diet therapy, all groups exhibited body mass gain, which varied significantly within the same group, except for the animals in the mdx + DCet group (p = 0.752 There was a significant difference in mass gain among the animals that received DC (p = 0.004) and among the mdx animals (p = 0.0001).

Although the mdx + DC group exhibited the lowest group final weight, the animals of this group showed the greatest individual mass gain (3.8 ± 3.00) during treatment, while the mdx + DCet animals maintained the mean mass gain during the 14 days diet with less variation (final - initial) comparing the groups (0.2 ± 3.49).

Among the wild animals group, it was observed that the ketogenic diet (wt + DCet) promoted mass gain compared to DC, without statistical differences (p = 0.059).

The mean feed intake and the daily caloric amount consumed are shown in Table 3.

Table 3

Descriptive data on food consumption and nutritional value per animal per day
Grupos	Consumo (g)	Valor calórico (kcal)	Carb (g)	Prot (g)	Lip (g)
wt + DC	4,4 ± 9,17	18	11,87	3,88	0,79
wt + DCet	2,2 ± 2,33	15	0,03	2,56	9,77
mdx + DC	3,5 ± 5,23	14	9,50	3,10	0,64
mdx + DCet	1,8 ± 1,57	12	0,02	2,19	8,37
Results presented as mean ± standard deviation or mean.

During the 14 days treatment, we could observe that the groups that received DC exhibited higher feed consumption when compared to those who received DCet, and the caloric consumption was proportional despite the higher energy density of DCet. Among the mdx animals, those fed with DC had the highest mean daily food intake (3.5 ± 5.23). The animals mdx + DCet had the lowest average daily consumption (1.8 ± 1.57) among the groups, with equivalent caloric intake.

MEMORY AND LEARNING

Memory and learning were assessed using the inhibitory avoidance test to assess aversive memory impairment (Fig. 1) and the open field habituation test to assess habituation memory (Fig. 2).

In the training session, there was no difference in latency time between the mdx groups and the wild (wt) groups. It was observed, however, that there was a statistically significant increase (p < 0.05) in latency between the test and the training of the wild animal groups and the mdx + DCet groups. The animals mdx + DC did not show significant differences in the time spent on the platform after 24 hours, suggesting a characteristic impairment of long-term aversive memory in this animal model. On the other hand, ketogenic diet treatment was significant in contributing to memory retention both in the animal model of the disease (mdx + DCet) and in the wild animals (wt + DCet), compared to the learning retained by the control group (wt + DC).

As for the open field habituation test, in the training session, there was no difference in latency time between the groups assessed. When comparing training with the test, there was a significant decrease in the number of crossings and rearings in the wt + DC and wt + DCet groups, evidencing retention of habituation memory. In the mdx + DC group, there was no significant decrease in memory. On the other hand, in the mdx animals submitted to ketogenic diet, a significant decrease in the number of crossings and rearings in the test session in relation to training can be observed, which means that there was no change in the habituation memory in these animals.

INFLAMMATORY PROCESS

The data from this session demonstrate the results obtained in the assessment of pro- and anti-inflammatory cytokine levels (Figs. 3 to 6) and MPO activity through Fig. 7.

Figure 3 shows the levels of TNF-α in the analyzed tissues: brain (3A

A significant increase in TNF-α levels was observed in the mdx + DC animals compared to the wild controls (wt + DC) specifically in the hippocampus regions (3C) and in the muscle tissue assessed (3F), where the increase was significant. However, with 14 days of ketogenic diet therapy (mdx + DCet) significant reductions (p < 0.05) were observed in the concentrations of this pro-inflammatory cytokine in the regions affected (3C and 3F). In the other regions assessed, there were no statistically significant changes between the groups.

Regarding the levels of IL-1β, another pro-inflammatory mediator, Fig. 4 shows the influence of diet therapy on the brain (4A – 4E) and muscle (4F) tissues per group of treated animals.

IL-1β levels were significantly elevated in mdx + DC animals compared to wt + DC animals in the prefrontal (4A), hippocampus (4C), cortex (4E) and gastrocnemius (4F) regions.).

As shown in Fig. 7, the quantification of IL-1β after 14 days of ketogenic diet indicated a significant reduction (p < 0.05) of this interleukin in the mdx + DCet group in the affected regions (4A, 4C, 4E, 4F) when compared to the animals mdx + DC. IL-1β levels were not altered in the cerebellum and striatum, suggesting that there was no significant inflammatory damage in these regions.

Figure 8 shows the analysis of IL-6 in the brain (5A – 5E) and muscle (5F) tissues of animals per diet per group.

The mdx + DC animals showed significantly lower levels of IL-6 (p < 0.05) in the hippocampus (5C) when compared with the wt + DC control animals.

After 14 days of treatment with a ketogenic diet, there was a significant increase in IL-6 levels in the hippocampus region (5C) of the mdx + DCet animals compared to mdx + DC animals. In the other areas assessed, including muscle tissue, there were no significant changes in this cytokine.

To assess the endogenous production of anti-inflammatory cytokines, the levels of IL-10, expressed in Fig. 6, were measured in the tissues analyzed: brain (6A–6E) and muscle (6F).

In the brain tissue, a significant decrease (p < 0.05) of IL-10 levels was observed in the mdx + DC animals compared to the control group (wt + DC) but only in the hippocampus (6C). IL-10 levels were also statistically lower in muscle tissue (6F) in the same group of untreated animals (mdx + DC).

On the other hand, only in the muscle tissue (6F) there was a significant recovery of the levels of this cytokine in mdx + DCet animals when compared with control animals in dx + DC. In the hippocampus, the increase in IL-10 levels by the ketogenic diet was not statistically significant, as well as in the other regions analyzed.

Figure 7 presents the result of MPO analysis in the brain (7A–7E) and muscle tissue (7F) by dietary therapy group.

Figure 8 shows the quantification of peroxidized lipids obtained in prefrontal cortex (8A), cerebellum (8B), hippocampus (8C), striatum (8D), cortex (8E) and gastrocnemius (8F).

The levels of lipid peroxidation were statistically (p < 0.05) higher in animals mdx (mdx + DC) compared to their control group (wt + DC) specifically in the regions of the hippocampus (8C), striatum (8D), cortex (8E) and muscle tissue (8F). In the regions C, E and F, treatment by the ketogenic diet promoted a significant reduction in MDA levels. In the striatum, diet therapy showed a reduction in lipid damage, but not significant. In the regions of the prefrontal cortex (8A) and cerebellum (8B), no statistical differences were observed between the wt and mdx groups.

Figure 9 shows the results of protein damage identified in prefrontal cortex (9A), cerebellum (9B), hippocampus (9C), striatum (9D), cortex (9E) and gastrocnemius (9F).

Oxidative damage in proteins showed significantly (p < 0.05) higher levels in the prefrontal cortex (9A), hippocampus (9C) and muscle tissue (9F) region of mdx + DC animals when compared to control animals (wt + DC). The ketogenic diet was effective to significantly reduce protein carbonylation in the hippocampus and muscle tissue (p < 0.05), while in the prefrontal region this reduction was not statistically significant. In the other structures (9B, 9D, 9E) there was no significant interference by the ketogenic diet between the wt and mdx groups.

Figure 10 shows the levels of free thiols obtained in the brain (10A – 10E) and muscular (10F) tissue of the assessed animals.

The figure shows that mdx animals fed a ketogenic diet (mdx + DCet) showed a significant increase (p < 0.05) in antioxidant activity when compared to mdx + DC animals specifically in the prefrontal cortex structures (10A), hippocampus (10C) and cortex (10E). In the other structures (10B, 10D) there was no significant interference by the ketogenic diet between the wt and mdx groups, including on the muscle tissue (10E). In all groups thiol protein level increased.

To assess the effects of the ketogenic diet on the initial stage of the respiratory chain, the activity of the respiratory complex I in brain tissue and gastrocnemius muscle (Fig. 11), in addition to creatine kinase (Fig. 12) in brain tissue and gastrocnemius muscle, were assessed.

Figure 11 shows the results of respiratory chain complex I activity on the prefrontal cortex (11A), cerebellum (11B), hippocampus (11C), striatum (11D), cortex (11E) and gastrocnemius (11F).

Figure 11 shows that among wild animals, only in the cerebellum (11B) there was a significant change (p < 0.05) between the type of intervention, with an increase in the enzymatic activity of complex I in the wt + DCet group. In this same region, it is observed that DCet influences enzymatic activity in both wt and mdx animals.

The mdx + DC animals showed significantly higher levels (p < 0.05) of complex I enzymatic activity in all analyzed tissues, with the exception of the striatum (11D) and the gastrocnemius muscle (11F). However, the intervention with the ketogenic diet promoted a significant reduction (p < 0.05) in the activity of this complex in mdx animals (mdx + DCet), with the exception of the striatum region (11D), where there was no significant change between the groups, showing that the protocol used was not able to interfere in this area assessed.

Figure 12 shows the results of CK activity in the prefrontal cortex (12A), cerebellum (12B), hippocampus (12C), striatum (12D), cortex (12E) and gastrocnemius (12F).

As seen in Fig. 12, a very heterogeneous CK activity was observed in the tissues assessed. In the cerebellum (12B) and cortex (12E) it was evident that the protocol used was not able to interfere in these regions of the CNS, with no significant changes in the parameter assessed.

However, mdx + DC mice exhibited greater (p < 0.05) CK activity than wt mice fed the same diet, in the prefrontal cortex (12A) and muscle tissue (12F) regions. In the striatum (12D), though, mdx mice regardless of diet showed a significant (p < 0.05) reduction in enzyme activity when compared to their controls. In the hippocampus (12C), this reduction was observed only in the mdx + DCet group, being significant (p < 0.05) when compared to the wt + DCet and mdx + DC groups.

Quantifications of BDNF in the brain tissue (Fig. 13A – 13E) per group of treated animals are described below.

In mdx + DC animals, BDNF levels were significantly lower (p < 0.05) in the hippocampus (13C) and in the striatum (13D) when compared to wt + DC control animals.

After 14 days treatment with a ketogenic diet, there was a significant increase in BDNF quantification in the hippocampal region (13C) of mdx + DCet animals compared to mdx + DC animals. The same difference was not significant in the striatum region. In the other regions assessed, there were no significant changes between the groups.

Although significant progress has been made in the scientific advancement of DMD, there is still no cure for the changes caused by this pathology. In this connection, our study shows that non-drug therapies, such as the ketogenic diet, which aim to minimize disease progression play a significant role in the treatment and improve the pathophysiology of DMD.

In this study, although the animals that received the ketogenic diet consumed a smaller amount of feed in grams, in kcal this consumption was little variable between the groups, since the energy density (kcal/g) of the ketogenic diet is higher compared to the control ration. A high-calorie diet influences appetite and consequently decreases food consumption[27, 28], observed in the wt + DCet and mdx + DCet groups.

It is important to emphasize that the relationship between energy consumption and body weight does not increase linearly with body weight increase [29]. In addition to this indirect relationship, the increase in energy consumption does not necessarily reflect an increase in adiposity due to a high-fat diet [30] thus, justifying the inverse proportion of high consumption and low mass gain represented by the wt + DC group.

The consequences for body composition also depend on the diet composition. Another factor of great importance for weight gain in wt + DCet, regardless of caloric intake, may have been the type of fat used in the diet. According to Buettner et al. (2007)[31], saturated and monounsaturated fat are capable of causing a more pronounced obesity and insulin resistance when compared to polyunsaturated fat, which are capable of generating an increase in lipogenesis and deposits of fat microvesicles in the liver[30].

Considering an altered metabolic state in DMD, we can assume that from these body mass and consumption results there is a greater metabolic demand in mdx animals, which can be supplied by the lipid source of energy, but not by the carbohydrates metabolic pathway. An important consideration already reported by Radley-Crabb et al (2011)[32] is that unless the baseline energy needs of DMD are met, the benefits of therapeutic interventions will not be fully obtained.

The results of the inflammatory process in this study corroborate the literature, showing a greater involvement in the skeletal muscle and in the CNS of the mdx mice and are supported by the findings of Comim et al. (2015) [33] who confirmed increased levels of IL-1β and TNF-α and reduced levels of IL-10 in the serum of young adult patients diagnosed with DMD thus showing a potent suppressive effect of the existing inflammatory process.

Also noteworthy is the fact that the hippocampus was the region most affected by changes in the cascade of inflammatory mediators, with significant changes in all cytokines assessed. Considering that the memory and learning processes are produced mainly in the hippocampus, and this brain region has a high density of receptors for IL-1, an effectiveness of the treatment with a ketogenic diet in this region during the neuroinflammatory process caused by DMD was found.

Pre-treatment with a ketogenic diet has already been associated with a reduction in the IL-1β response in the hippocampus four hours after LPS injection in Wistar rats [14]. In contrast, TNF-α levels were not different after pre-treatment with a ketogenic diet, supplemented or not with omega 3, in the regions of the hippocampus[14, 34] and striatum[33] in Wistar rats fed with a ketogenic diet. However, at serum levels, Paoli et al. (2015) [35] found significant reductions in TNF-α levels in overweight subjects who followed a four-week ketogenic diet protocol without polyunsaturated fatty acid supplementation. The differences found may be related to the different time periods of diet administration, ranging from 14 days to 8 weeks[34].

The main findings of the present study are: (1) first evidence that the ketogenic diet improves memory and behavior in mdx mice; (2) the neuroprotective effects of the ketogenic diet are mediated by a reduction in cytokine and expression; (3) both inflammation and OS are associated with memory consolidation in the mdx + DCet model; and, (4) the ketogenic diet exerts important effects on memory and learning, but does not prevent the inflammatory and oxidative process of DMD.

Taken together, these findings suggest that the ketogenic diet can modulate the CNS adaptive responses through the cognitive performance in the DMD animal model. Although further investigation into the underlying mechanisms of action is needed, the evidence available suggests that the ketogenic diet is a regimen that deserves attention as a potential primary therapy for neurodegenerative diseases, especially DMD.

Author Contribution: Lilian Leite Fausto, Viviane Freiberger, and Letícia Ventura planned the experiments and wrote the paper. Clarissa Martinelli Comim and Adriano Alberti managed the project and performed most of the experiments and analysis and provided input on statistical analysis and interpretation. Adriano Alberti provided substantial input for the manuscript and wrote the paper. Daniel Fernandes Martins, Gabriela Kades, Risoni Dias, Paula Dias, Eliton Marcio Zanoni, Bem Hur Soares and Matheus Luchini Dutra provided technical guidance and supervision. All authors read and approved the fnal manuscript.

Funding This research was supported by grants from Unisul (CMC) and the CAPES (LLF).

Data Availability Data will be made available upon request.

Acknowledgements I thank the funding sources of this study and the entire group for their dedication.

Conflict of Interest The authors declare no competing interests.

Consent to Participate Not applicable.

Consent for Publication Not applicable

Chung J, Smith AL, Hughes SC, Niizawa G, Abdel-Hamid HZ, Naylor EW, et al. Twenty-year follow-up of newborn screening for patients with muscular dystrophy. Muscle Nerve. abril de 2016;53(4):570–8.
Nocetti PM, Alberti A, Freiberger V, Ventura L, Grigollo LR, Andreau CS, et al. Swimming Improves Memory and Antioxidant Defense in an Animal Model of Duchenne Muscular Dystrophy. Mol Neurobiol . 2021;58(10):5067–77.
Moser H. Duchenne muscular dystrophy: pathogenetic aspects and genetic prevention. Hum Genet. 1984;66(1):17–40.
Falzarano MS, Scotton C, Passarelli C, Ferlini A. Duchenne muscular dystrophy: From diagnosis to therapy. Molecules. 2015;20(10):18168–84.
Wagner KR, Lechtzin N, Judge DP. Current treatment of adult Duchenne muscular dystrophy. Biochim Biophys Acta. 2007;1772(2):229–37.
Wang H-S, Lin K-L. Ketogenic diet: an early option for epilepsy treatment, instead of a last choice only. Biomed J. 2013;36(1):16–7.
Misiewicz Runyon A, So T-Y. The Use of Ketogenic Diet in Pediatric Patients with Epilepsy. ISRN Pediatr. 2012;2012:1–10.
Puchowicz MA, Zechel JL, Valerio J, Emancipator DS, Xu K, Pundik S, et al. Neuroprotection in diet-induced ketotic rat brain after focal ischemia. J Cereb Blood Flow Metab. 23 de dezembro de 2008;28(12):1907–16.
Hallböök T, Ji S, Maudsley S, Martin B. The effects of the ketogenic diet on behavior and cognition. Epilepsy Res. julho de 2012;100(3):304–9.
David N. Ruskin, Tracey A.C.S. Suter, Jessica L. Ross and SAM. Ketogenic diets and thermal pain: dissociation of hypoalgesia, elevated ketones, and lowered glucose in rats. J Pain. 2013;100(2):130–4.
Jarrett SG, Milder JB, Liang LP, Patel M. The ketogenic diet increases mitochondrial glutathione levels. J Neurochem. 2008;106(3):1044–51.
Ahola-Erkkilä S, Carroll CJ, Peltola-Mjösund K, Tulkki V, Mattila I, Seppänen- Laakso T, et al. Ketogenic diet slows down mitochondrial myopathy progression in mice. Hum Mol Genet. 2010;19(10):1974–84.
Kristopher Bough P. Energy Metabolism as Part of the Anticonvuslant Mechanism of the Ketogenic Diet. Epilepsia 2008 Novemb ; 49(Suppl 8) 91–93 doi101111/j1528-1167200801846.x Energy. 2008;49(Suppl 8):91–3.
Dupuis N, Curatolo N, Benoist JF, Auvin S. Ketogenic diet exhibits anti- inflammatory properties. Epilepsia. 2015;56(7):e95–8.
Napoli E, Dueñas N, Giulivi C. Potential therapeutic use of the ketogenic diet in autism spectrum disorders. Front Pediatr. 2014;2(June):69.
Dutton SBB, Sawyer NT, Kalume F, Jumbo-Lucioni P, Borges K, Catterall WA, et al. Protective effect of the ketogenic diet in Scn1a mutant mice. Epilepsia. 2011;52(11):2050–6.
Newell C, Bomhof MR, Reimer RA, Hittel DS, Rho JM, Shearer J. Ketogenic diet modifies the gut microbiota in a murine model of autism spectrum disorder. Mol Autism. 2016;7(1):37.
Tolazzi J da R, Doile RG, Bezerra AS. Nutrição Experimental : Conceitos ,. Discip Sci. 2015;16:147–62.
Cancelliero KM, Costa D, Silva CA da. Isolamento social modifica o perfil bioquímico de ratos. Rev bras Zoociências. 2005;247–57.
Quevedo J, Vianna MR, Roesler R, De-Paris F, Izquierdo I, Rose SP. Two time windows of anisomycin-induced amnesia for inhibitory avoidance training in rats: protection from amnesia by pretraining but not pre-exposure to the task apparatus. Learn Mem. 6(6):600–7.
Anderson JL, Head SI, Rae C, Morley JW. Brain function in Duchenne muscular dystrophy. Brain. 2002;125(Pt 1):4–13.
Draper HH, Hadley M. Malondialdehyde determination as index of lipid peroxidation. Methods Enzymol. 1990;186:421–31.
Levine RL, Garland D, Oliver CN, Amici A, Climent I, Lenz AG, et al. Determination of carbonyl content in oxidatively modified proteins. Methods Enzymol. 1990;186:464–78.
Puhl H, Waeg G, Esterbauer H. Methods to determine oxidation of low-density lipoproteins. Methods Enzymol. 1994;233:425–41.
Cassina A, Radi R. Differential Inhibitory Action of Nitric Oxide and Peroxynitrite on Mitochondrial Electron Transport. Arch Biochem Biophys. abril de 1996;328(2):309–16.
HUGHES BP. A method for the estimation of serum creatine kinase and its use in comparing creatine kinase and aldolase activity in normal and pathological sera. Clin Chim Acta. setembro de 1962;7:597–603.
Dos Santos B, Estadella D, Hachul ACL, Okuda MH, Moreno MF, Oyama LM, et al. Effects of a diet enriched with polyunsaturated, saturated, or trans fatty acids on cytokine content in the liver, white adipose tissue, and skeletal muscle of adult mice. Mediators Inflamm. 2013;2013.
Ribeiro G, Santos O. Recompensa alimentar: mecanismos envolvidos e implicações para a obesidade. Rev Port Endocrinol Diabetes e Metab. 2013;8(2):82–8.
Faria HG de. Considerações sobre dietas experimentais para animais de laboratório: formulações, aplicações, fornecimento e efeitos experimentais. In: I Simpósio de bioterismo da Fiocruz - PE. Recife: Fiocruz; 2010. p. 16.
White PAS, Cercato LM, Araújo JMD, Souza LA, Soares AF, Barbosa APO, et al. Modelo de obesidade induzida por dieta hiperlipídica e associada à resistência à ação da insulina e intolerância à glicose. Arq Bras Endocrinol Metabol. 2013;57(5):339–45.
Buettner R, Scholmerich J, Bollheimer LC. High-fat diets: Modeling the metabolic disorders of human obesity in rodents. Obesity. 2007;15(4):798–808.
Radley-Crabb HG, Fiorotto ML, Grounds MD. The different impact of a high fat diet on dystrophic mdx and control C57Bl/10 mice. PLoS Curr. 15 de novembro de 2011;3:RRN1276.
Comim CM, Mathia GB, Hoepers A, Tuon L, Kapczinski F, Dal-Pizzol F, et al. Neurotrophins, cytokines, oxidative parameters and funcionality in Progressive Muscular Dystrophies. An Acad Bras Cienc. 2015;87(3):1809–18.
Vizuete AF, De Souza DF, Guerra MC, Batassini C, Dutra MF, Bernardi C, et al. Brain changes in BDNF and S100B induced by ketogenic diets in Wistar rats. Life Sci. 2013;92(17–19):923–8.
Paoli A, Moro T, Bosco G, Bianco A, Grimaldi KA, Camporesi E, et al. Effects of n-3 polyunsaturated fatty acids (??-3) supplementation on some cardiovascular risk factors with a ketogenic mediterranean diet. Mar Drugs. 2015;13(2):996–1009.

Download PDF

Version 1

posted

You are reading this latest preprint version

Effects of a Ketogenic Diet on the Evaluation of Biochemical and Clinical Parameters of Duchenne Muscular Dystrophy: a Preclinical Study

Status:

Version 1

Abstract

Figures

Introduction

Materials And Methods

Results

BODY MASS AND FEED CONSUMPTION

MEMORY AND LEARNING

INFLAMMATORY PROCESS

Discussion

Conclusion

Declarations

References

Status:

Version 1