Dietary Omega-3 Fatty Acids Do Not Change Resistance of Rat Brain or Liver Mitochondria to Ca2+ and/or Prooxidants

Omega-3 polyunsaturated fatty acids (n-3 PUFAs) block apoptotic neuronal cell death and are strongly neuroprotective in acute and chronic neurodegeneration. Theoretical considerations, indirect data, and consideration of parsimony lead to the hypothesis that modulation of mitochondrial pathway(s) underlies at least some of the neuroprotective effects of n-3 PUFAs. We therefore systematically tested this hypothesis on healthy male FBFN1 rats fed for four weeks with isocaloric, 10% fat-containing diets supplemented with 1, 3, or 10% fish oil (FO). High resolution mass spectrometric analysis confirmed expected diet-driven increases in docosahexaenoic acid (DHA, 22:6, n-3) and eicosapentaenoic acid (EPA, 20:5, n-3) in sera, liver and nonsynaptosomal brain mitochondria. We further evaluated the resistance of brain and liver mitochondria to Ca2+ overload and prooxidants. Under these conditions, neither mitochondrial resistance to Ca2+ overload and prooxidants nor mitochondrial physiology is altered by diet, despite the expected incorporation of DHA and EPA in mitochondrial membranes and plasma. Collectively, the data eliminate one of the previously proposed mechanism(s) that n-3 PUFA induced augmentation of mitochondrial resistance to the oxidant/calcium-driven dysfunction. These data furthermore allow us to define a specific series of follow-up experiments to test related hypotheses about the effect of n-3 PUFAs on brain mitochondria.


INTRODUCTION: Background
a. Include sufficient scientific background (including relevant references to previous work) to understand the motivation and context for the study, and explain the experimental approach and rationale.
In mammals, the central nervous system (CNS) has the second highest concentration of lipids after adipose tissue. Lipids play a critical role in neuronal membrane function, as well as in enzyme, receptor and ion channel activities [1,6]. One of the main constituents of brain phospholipids is the omega-3 group of polyunsaturated fatty acids (n-3 PUFAs). There are three major n-3 PUFAs: alpha-linolenic (ALA), eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids. DHA is an essential n-3 PUFA for brain. It is highly enriched in neural membranes, constituting 30-40% of phospholipids in the cerebral cortex and retina [9,20]. Because brain tissue is unable to make n-3 PUFAs, it is remarkably sensitive to adequate diet supplementation during all stages of CNS development -from embryonic differentiation to adulthood and aging [6,20,22,43,51]. Neural trauma and neurodegeneration are associated with significant disturbances in neuronal membrane phospholipid metabolism [13,28,36], suggesting that supplementation with n-3 PUFAs may be beneficial for recovery.
Omega-3 deficiency induces structural and functional abnormalities in the hippocampus, hypothalamus and cortex -brain areas that mediate spatial and serial learning [51]. Omega-3 deficiency significantly reduces the level of cerebral catecholamines, brain glucose transport capacity and glucose utilization, cyclic AMP level, and the capacity for phospholipid synthesis. Such a deficiency also markedly affects neuronal membrane fluidity, activity of membrane-bound enzymes, receptors and ion channels (e.g., Na + ,K + -ATPase), production of neurotransmitters and brain peptides, gene expression (such as peroxisome prolifirator-activated receptor α (PPAR-α), sterol regulatory binding protein-1 (SREBP-1), carbohydrate regulatory element binding protein/Max-like factor X heterodimer (CREBP/Max-like factor X) [21], intensity of inflammation and synaptic plasticity [1,7,11,51].
Within the cell, the mitochondrial membrane is a primary site for n-3 PUFA incorporation [17,19,38,40,48]. A growing body of evidence has established that mitochondria are a key component in the signaling pathway(s) underlying cell death [2,14,15,29,44,45,47]. To some extent, mitochondria serve to integrate different apoptosis-inducing stimuli (Ca 2+ , proapoptotic Bcl-2 family proteins, reactive oxygen species, etc.) and direct them into a common downstream pathway [2,14,29,45]. Mitochondria are enlisted to initiate the downstream stages of cell death through mitochondria permeability transition (MPT)-dependent and independent mechanisms. The MPT is a multiprotein pore complex of as yet unidentified structure that is presumably localized at the contact sites between the inner and outer mitochondrial membranes. The MPT begins as a permeabilization of the inner membrane, which prevents buildup of a mitochondrial membrane potential, and leads to loss of the ability to sequester calcium from the medium, progressive osmotic swelling, disruption of the outer membrane, loss of matrix and intermembrane proteins, and initiation of caspasedependent and caspase-independent cell death pathways [2,29]. Mitochondrial damage, occurring via the MPT, has been identified as a critical event in stroke and stroke-related injuries, secondary injury following brain trauma (TBI), and chronic neurodegeneration [2,15,29,45,47].
In light of the aforementioned links between mitochondria and cell death, mitochondria and n-3 PUFAs, and n-3 PUFAs and neuronal function, it is noteworthy that recent evidence shows that omega-3 PUFAs can modulate processes that contribute to the secondary degeneration of the CNS [18,27,36,49,50]. Particularly, administration of n-3 PUFAs after spinal cord compression injury in rats significantly increased neuronal survival and improved locomotive performance for up to 6 weeks after injury. Furthermore, pre-injury diet supplementation with omega-3 PUFAs prevented some TBI-induced effects -a reduction in synaptic plasticity, impaired learning ability, oxidative damage. Recent data suggest that eight weeks of dietary supplementation with DHA prevents induction of MPT in cardiac mitochondria [23]. These data, coupled with the above background, suggests that diets enriched in n-3 PUFAs affect mitochondria in a way that makes them more resistant to the oxidant-and calcium-mediated injury associated with both acute neurological injury and induction of the MPT.
The goal of present study was, therefore, to test directly the involvement of the MPT pathway in n-3 PUFAmediated neuroprotection. Specifically, we determined whether 4 weeks dietary supplementation in rats with 1, 3 or 10% fish oil (FO) containing essential n-3 PUFAs, e.g., EPA and DHA, changes the resistance of isolated nonsynaptosomal brain and liver mitochondria to proapoptotic signals such as Cа 2+ and prooxidants.
b. Explain how and why the animal species and model being used can address the scientific objectives and, where appropriate, the study's relevance to human biology.
The centerpiece of the study was the evaluation of dietary ώ-3 PUFA mediated effect on mitochondrial function isolated from brain and liver tissues. We have utilized animals, in a contrast, cell cultures, for instance, because they are the only model that can test the overall effects of diet on the organism including tissue specificity. Rats were chosen as they are well-characterized animal models of both diet and mitochondrial function, and they are the phylogenetically the lowest animal species that provided sufficient material to complete the planned experiments.

4
Clearly describe the primary and any secondary objectives of the study, or specific hypotheses being tested.
The objective of the study was to test a specific hypothesis -whether diets enriched in n-3 PUFAs affect mitochondria in a way that makes them more resistant to oxidant-and calcium-mediated injury. There are 4 groups of 24 animals each. There is one control group fed with control (w/o fish oil) diet, and the three experimental groups each fed with 1, 3 or 10% fish oil diets.

b.
Any steps taken to minimize the effects of subjective bias when allocating animals to treatment (e.g. randomization procedure) and when assessing results (e.g. if done, describe who was blinded and when).
We studied four diets simultaneously. The animals were ordered from Harlan to be approximately 3 weeks of age (20-25 days) and of similar body weight. The animals were assigned to cages randomly by the husbandry staff at unpacking and then the diets were assigned to each cage (2 animals per cage) randomly. c.

The experimental unit (e.g. a single animal, group or cage of animals). A time-line diagram or flow chart can be useful to illustrate how complex study designs were carried out.
The experimental unit for isolation of brain mitochondria is four animals (or, more specifically, brain tissues extracted from four animals were combined to isolate brain mitochondria). The experimental unit for isolation of liver mitochondria is one animal. Animals were always done by dietary group, e.g., four animals from the diet currently used.

7
For each experiment and each experimental group, including controls, provide precise details of all procedures carried out. For example:

a. How (e.g. drug formulation and dose, site and route of administration, anesthesia and analgesia used [including monitoring], surgical procedure, method of euthanasia). Provide details of any specialist equipment used, including supplier(s).
No drugs and no specialist equipment. Complete dietary information is provided in this appendix. .
Animal colony procedure room d. Why (e.g. rationale for choice of specific anesthetic, route of administration, drug dose used).

NA
Sacrifice by decapitation without anesthesia following IACUC approved exemption due to known interference with assays. 8 a. Provide details of the animals used, including species, strain, sex, developmental stage (e.g. mean or median age plus age range) and weight (e.g. mean or median weight plus weight range).

EXPERIMENTAL: Experimental animals
We used FBNF1 male rats. Animals were brought into the colony at approximately 3 weeks of age and then maintained for 5 weeks on their assigned diet prior to sacrifice. The mean weight growth chart is presented below as Mean ± SD. The animals were FBNF1 male rats acquired from Harlan Laboratories at approximately 3 weeks of age and of similar body weight. The diets were purchased from Research Diets, Inc. The animals were maintained in a nonbarrier animal facility operated by Harvard Medical School. The animal facility is a non-barrier animal facility operated by Harvard Medical School. The animals were housed 2 per cage. The cages are standard plastic rat cages with wire lids (to hold food and water) and plastic, filtered tops. The animals live on the solid cage bottom with wood chip bedding (alpha chip). Cages are changed at least twice per week.

b.
Husbandry conditions (e.g. breeding programme, light/dark cycle, temperature, quality of water etc for fish, type of food, access to food and water, environmental enrichment).
The light/dark cycle is 12 hour/12 hour. The temperature and humidity are maintained at 69 degrees and 45% respectively. The animals have ad lib access to food and water.

c.
Welfare-related assessments and interventions that were carried out prior to, during, or after the experiment.
All work with animals must be approved by the IACUC committee at Brigham and Women's Hospital. Harvard Medical School has a full time staff of veterinarians and veterinary technicians that visit the facility on a regular weekly schedule. A veterinarian is on call during evenings, weekends, and holidays. The husbandry crew is present in the facility every day including weekends and holidays.
The animals on this study were not expected to suffer any ill effects clinically. And as expected, no animal appeared to be anything less than healthy at any time. 10 a. Specify the total number of animals used in each experiment, and the number of animals in each experimental group.

EXPERIMENTAL: Sample size
There are 4 groups of 24 animals each. Four animals from one dietary group were sacrificed at a time to obtain a sufficient yield of non-synaptosomal brain mitochondria. The liver tissue was extracted from only one animal (from the one of four animals used for brain extraction). The control group of animals was fed with control (w/o fish oil) diet and the three experimental animal groups were fed with one of either 1, 3 or 10% fish oil diet.

b.
Explain how the number of animals was arrived at. Provide details of any sample size calculation used.
From experience it was estimated that this N (N=6) was sufficient to determine a trend of the dietarymediated effect and conclude whether it is worth to continue the experiments in this direction. c.
Indicate the number of independent replications of each experiment, if relevant.
The experiments were replicated 6 times.

EXPERIMENTAL: Allocating animals to experimental groups
11 a. Give full details of how animals were allocated to experimental groups, including randomization or matching if done.
The diets were chosen in advance of the animals arriving. The animals were randomly assigned to cages, 2 per cage, by the husbandry crew at arrival. The cages were randomly assigned a diet.
b. Describe the order in which the animals in the different experimental groups were treated and assessed.
The experimental unit is one animal (or, more specifically, liver mitochondria isolated from one animal) and four animals for non-synaptosomal brain mitochondria isolated from four animals. Four animals from one dietary group were used for each experiment.

12
Clearly define the primary and secondary experimental outcomes assessed (e.g. cell death, molecular markers, behavioural changes).
The study design was focused to link dietary fish oil to changes in mitochondrial sensitivity to proapoptotic agents (e.g., calcium and prooxidants). It also tested the incorporation of n-3 polyunsaturated fatty acids in mitochondrial membrane to link the changes in membrane composition to function.