Forebrain cellular bioenergetics in neonatal mice

Abstract

BACKGROUND:

Hypoglycemia occurs frequently in the neonate and may result in neurologic dysfunction. Its impact on the kinetics of cellular respiration and bioenergetics in the neonatal brain remains to be explored.

AIMS:

Develop murine model to investigate the effects of hypoglycemia on neonatal brain bioenergetics.

STUDY DESIGN:

Forebrain fragments were excised from euthanized BALB/c pups aged <24 hours to 14 days. We measured cellular respiration (μM O₂ min^–1.mg^–1) in phosphate-buffered saline with and without glucose, using phosphorescence oxygen analyzer, as well as cellular adenosine triphosphate (ATP, nmol.mg^–1) using the luciferin-luciferase system.

RESULTS:

In the presence of glucose, although cellular respiration was 11% lower in pups ≤3 days compared to those 3– 14 days old (0.48 vs. 0.54), that difference was not statistically significant (p = 0.14). Respiration driven by endogenous metabolic fuels (without added glucose) was 16% lower in pups ≤3 days compared to those 3– 14 days (0.35 vs. 0.42, p = 0.03), confirming their increased dependency on exogenous glucose. Although cellular ATP was similar between the two age groups (14.9 vs. 11.2, p = 0.32), the ATP content was more severely depleted without added glucose in the younger pups, especially in the presence of the cytochrome c oxidase inhibitor cyanide. The first-order rate constant of cellular ATP decay (hydrolysis) was 44% lower in 2-day-old pups compared to 14-day-old mice (0.43 vs. 0.77 min^–1, p = 0.03).

CONCLUSIONS:

Forebrain cellular respiration and ATP consumption are lower in young pups than older mice. In the absence of glucose, the support for these processes is reduced in young pups, explaining their brain hypersensitivity to hypoglycemia.

Keywords

Hypoglycemia,neonates cellular respiration cellular bioenergetics

1 Introduction

The brain consumes 60% of all utilized glucose by the body, making this nutrient the main metabolic fuel to generate adenosine triphosphate (ATP) for physiologic brain function. As neurons have the highest energy demand in the body, the brain requires continuous delivery of glucose [1], and cannot survive more than a few minutes of glucose deprivation [2]. Episodes of hypoglycemia compromise brain structure [1], and function, leading to long-term neurodevelopmental sequelae [2].

Hypoglycemia is common during the neonatal metabolic transition to the extra uterine environment [3, 4], especially in preterm, growth restricted newborn, or infants of diabetic mothers. The hypoglycemia-induced cerebral injury may be sub-clinical, or presents as neurologic dysfunctions, such as irritability, neurological deficits, seizure, coma and, ultimately, neuronal death with permanent long-term neurodevelopmental impairment [5 –7]. Most neonates with severe hypoglycemia also have white matter abnormalities on brain magnetic resonance imaging [8].

The mechanisms contributing to hypoglycemic brain injury and their relationships with the severity and duration of hypoglycemia in neonates remain to be elucidated. The immature brain is more sensitive to limitations of substrate availability because of minimal high-energy phosphates cerebral reserves [9, 10]. This might explain the vulnerability of the neonatal brain to hypoglycemia induced injury. Although glucose is the primary fuel for cerebral oxidative metabolism, the physiological metabolic adaptation to postnatal life also involves the use of lactate [11, 12], and ketone bodies [3], as alternative substrates for oxidative metabolism. The preterm infant, however, has a limited ability to mobilize these nutrients [3].

The mitochondrial respiratory chain is the focal point for energy metabolism in the brain, since it links substrate (such as glucose) and O₂ consumption with ATP production. Mitochondria are essential for the functioning of the neurons because their limited glycolytic capacity makes their energetic needs highly dependent on oxidative phosphorylation [13]. This is reflected by the high oxygen demand of central nervous system, which, in the human newborn, accounts for approximately 10% of body weight, but disproportionally utilizes 20% of all inspired oxygen [14]. Most of the ATP required for the normal functioning of neurons is generated by oxidative phosphorylation [15, 16]. Although the brain contains glycogen [17], its complete oxidation would sustain basal respiration for only 20 minutes and would be quickly depleted without the prior availability of external glucose [18]. Glycolysis is, therefore, highly dependent on external glucose with an existing tight coupling between cellular respiration and glycolytic rate. During hypoglycemia, the decreased rate of glycolysis results in a decrease in the oxidative phosphorylation and also in the levels of cerebral high-energy phosphates; this may lead to altered neuronal and glial function and, eventually, cell death [19, 20].

As far as we know, no studies exist regarding the effects of hypoglycemia on murine neonatal brain cellular O₂ consumption and ATP content. As the majority of previous experimental studies were performed on chemically induced diabetes in mature animals that were administered insulin, the role of the preceding period of hyperglycemia and the possible role of insulin of the cellular mechanisms are likely to have confounded the role of hypoglycemia on the observed results [21 –25]. We therefore developed a novel neonatal murine model to analyze the in-vitro effects of hypoglycemia on brain cellular respiration and bioenergetics, without insulin-induced hypoglycemia. Mitochondrial O₂ consumption (cellular respiration) and cellular bioenergetics (cellular ATP) were considered surrogate biomarkers for the hypoglycemia-induced brain injury. The study hypothesis is that hypoglycemia-induced brain insult results from depletion of cellular bioenergetics.

2 Methods

2.1 Principles

We developed an in-vitro system, based on measurements of O₂ consumption by murine brain tissue, which could be utilized for accurate monitoring of hypoglycemia-induced impairments of cellular respiration and cellular bioenergetics. Cellular respiration is the process of delivering nutrients and O₂ to the mitochondria, oxidation of reduced metabolic fuels, passage of electrons to O₂, and synthesis of ATP. In this study, the effects of hypoglycemia on cellular respiration and ATP content were investigated in neonatal mice.

The principle underpinning this project is that cells consume O₂ at a constant rate (zero-order kinetics). Thus, in vessels sealed from air, O₂ concentration in solutions containing tissue fragments and glucose (as a respiratory substrate) declines linearly with time. Therefore, the rate of respiration is the negative of the slope of a plot of [O₂] vs. time. This rate and the pattern of the decrease in cellular O₂ consumption with time are analyzed in this study. Without adding glucose, endogenous metabolic fuels are gradually depleted with time, halting cellular respiration. The rate of O₂ consumption decreases with consumption of the substrates for glycolysis before respiration stops altogether. The addition of glucose oxidase (catalyzes the reaction D-glucose + O₂ ⟶ D-glucono-δ-lactone + H₂O₂) depletes residual glucose in the interstitium, allowing accurate monitoring of cellular bioenergetics in complete absence of glucose. Cellular respiration and ATP synthesis are inhibited by the addition of cyanide, which confirm these vital processes occur in the mitochondrial respiratory chain.

2.2 Ethical approval

The project was approved by the United Arab Emirates University Animal Ethics Committee (ERA_2016_4247). All procedures performed in studies involving animals were in accordance with the ethical standards of the United Arab Emirates University where the studies were conducted.

2.3 Animals

BALB/c pups (from <24 h to 14 days of age) bred in our institution’s animal facility were used for each experiment. Each mouse was used only once. Immediately before each experiment, euthanasia was performed on the animals, as per our institution’s animal laboratory policies and guidelines. A punch biopsy of the frontal lobe was immediately performed while the mouse’s heart was still beating. The biopsied specimen typically weighed less than 10 mg.

2.4 Measurement of O₂ consumption

The phosphorescence oxygen analyzer that measures dissolved O₂ concentration ([O₂]) in solution as a function of time was used to determine the rate of cellular respiration [26, 27]. This method is based on the principle that O₂ quenches the phosphorescence of a palladium (Pd) phosphor. The Pd (II) derivative of meso-tetra-(4-sulfonatophenyl)-tetrabenzoporphyrin (with an absorption maximum at 625 nm and a phosphorescence emission maximum at 800 nm) is used in this study for this purpose.

Forebrain fragments were excised and immediately placed in vials containing 1-ml phosphate-buffered saline (PBS), 3μM Pd phosphor, and 0.5% fat-free albumin for the O₂ measurement at 37°C. The Pd phosphor solution was prepared daily, kept on ice, and warmed to 37°C prior to use. The vials were sealed with crimp top aluminum seals. Mixing was performed with the aid of parylene-coated stirring bars.

Samples were exposed to light flashes (10 per sec) from a pulsed light-emitting diode array with a peak output at 625 nm. Emitted phosphorescent light was detected by a Hamamatsu photomultiplier tube after first passing it through a wide-band interference filter centered at 800 nm. Amplified phosphorescence was digitized at 1–2 MHz using an analog/digital converter (PCI-DAS 4020/12 I/O Board) with 1 to 20 MHz outputs.

The phosphorescence decay rate (1/τ) was characterized by a single exponential; I = Ae^–t/τ, where I = Pd phosphor phosphorescence intensity. The values of 1/τ are linear with dissolved O₂: 1/τ= 1/τ _o + k _q[O₂], where 1/τ= the phosphorescence decay rate in the presence of O₂, 1/τ _o = the phosphorescence decay rate in the absence of O₂, and k _q = the second-order O₂ quenching rate constant in sec^–1μM^–1.

A software package developed using Microsoft Visual Basic 6, Microsoft Access Database 2007, and Universal Library components (Universal Library for Measurements Computing Devices, http://www.mccdaq.com/daq-software/universal-library.aspx) was used for the analysis. It allowed direct reading from the PCI-DAS 4020/12 I/O Board (PCI-DAS 4020/12 I/O Board, http://www.mccdaq.com/pci-data-acquisition/PCI-DAS4020-12.aspx). The software included relational databases that stores experiments, pulses, and pulse metadata, including slopes. Pulse detection was accomplished by searching for 10 phosphorescence intensities greater than 1.0 V (by default). Peak detection was accomplished by searching for the highest 10 data points of a pulse and choosing the data point closest to the pulse decay curve from the 10 highest data points of a pulse. Depending on the sample rate, a minimum number of data points per pulse was set and used as a cutoff to remove invalid pulses with too few data points [27]. The results of O₂ consumption were expressed as μM O₂ min^–1 mg^–1 brain tissue.

2.5 Measurement of cellular ATP

Small forebrain fragments (<10 mg) were immer-sed in 1.0 mL ice-cold 2% trichloroacetic acid (prepared daily) and rapidly homogenized by vigorous vortexing. The samples were then centrifuged at –8°C at 16,873 g for 10 min, and the supernatants were stored at –20°C until analysis. For ATP determination, 50μL of the cellular acid extracts were thawed on ice and neutralized with 50μL of 100 mM Tris-acetate and 2 mM ethylene diamine tetra acetic acid (pH 7.75). The measurements used the Enliten ATP Assay System (Bioluminescence Detection Kit, Promega, Madison, WI). The luminescence reaction contained 5μL of neutralized acid-soluble supernatant and 45μL of luciferin/luciferase reagent. The luminescence intensity was measured at 25°C using the Glomax Luminometer (Promega, Madison, WI). ATP contents were expressed in nmol/mg protein (using the Bio-Rad Protein Assay Cat. #500-0006, Bio-Rad, Hercules, California, USA).

2.6 Analysis

Data analysis was performed with Stata version 13 (Stata Corp, TX, USA). Kaleida Graph version 4.5 (Synergy Software, PA, USA) was used for curve fitting. Standard descriptive analysis was performed, the Student t test was used to compare the values between two groups and analysis of variance (ANOVA) when comparing three groups. A 2-tailed p-value of 0.05 defined statistical significance.

3 Results

3.1 Descriptive analysis

Four age groups of pups were used initially for all the experiments: [1] ≤24 h (n = 22, median weight 2 g, weight range 1.3–2.2 g), [2] one to 3 days (n = 15, median weight 3 g, weight range 2.2–3.9 g), [3] 3 to 7 days (n = 10, median weight 5 g, weight range 4.2–6.5 g), and [4] 7 to 14 days (n = 16, median weight 5.6 g, weight range 4.9–6.2 g). As there was no significant difference in O₂ consumption between the first two groups nor between the last two (results not shown, p > 0.05), we therefore analyzed the results as two groups: [A] ≤3 days (n = 37) and [B] 3–14 days (n = 26).

3.2 Proof of concept validity

The brain tissue suspended in PBS alone (without glucose) had a steady rate of O₂ consumption for several minutes before its rate dropped, reflecting sufficient endogenous metabolic fuels to drive cellular respiration for a few minutes, followed by a cessation of respiration (Fig. 1A). The addition of cyanide inhibited O₂ consumption (Fig. 1B), confirming that O₂ was consumed in the mitochondrial respiratory chain. The lack of a response to glucose oxidase (before the addition of glucose) confirmed the respiration was driven by the endogenous respiratory fuels. These results support the validity of our assay that measures the impact of hypoglycemia on cellular respiration.

Fig.1

Representative runs of forebrain cellular respiration. Panel a. First run: Forebrain specimen was collected from the studied mouse and processed immediately for measuring cellular mitochondrial O₂ consumption in phosphate-buffered saline (PBS) with 0.1 mg/mL glucose oxidase. The initial rate of respiration (k, in μM O₂ min^–1) was set as the slope of the first run (linear fit with R² ≥0.970). The rate was then corrected for the specimen weight and expressed as k_c in μM O₂ min^–1 mg^–1. Second run: At the end of the first run, the specimen was transferred to a new O₂ vial that contained PBS with glucose oxidase and the measurement of cellular respiration was repeated. The lower value of k_c reflected partial depletion of the endogenous metabolic fuels. Third run: The same process was repeated to illustrate the complete depletion endogenous metabolic fuels in the absence of adding glucose to the solution. Panel b. Forebrain cellular respiration showing inhibition by sodium cyanide, which confirmed oxygen was consumed in the mitochondrial respiratory chain. The brain tissue had adequate endogenous fuels to drive cellular respiration for several minutes. O₂ concentration did not drop after the addition of glucose oxidase (catalyzes the reaction D-glucose + O₂ ⟶ D-glucono-δ-lactone + H₂O₂), confirming lack of glucose in the solution and the respiration was driven only be the endogenous metabolic fuels. O₂ was then depleted after the addition of glucose.

O₂ consumption by the brain tissue (mean k_c = 0.53μM O₂ min^–1 mg^–1, SD 1.4) was significantly higher (p < 0.05) than that of other tissues (kidney 0.34, liver 0.27, lung 0.24, pancreas 0.35, and spleen 0.28μM O₂ min^–1 mg^–1) in the same animal model [26]. These results are consistent with the higher O₂ utilization by the brain and confirm the validity of this experimental model.

3.3 Oxygen consumption

There was a more significant decrease in the rate of O₂ consumption without glucose than with added glucose in pups younger than three days (p < 0.001) compared to mice older than 3 days (p = 0.02). In the presence of glucose, there was no significant increase in glucose-supported respiration with age (p = 0.14), but cellular respiration driven by the endogenous metabolic fuels (without glucose) was significantly lower in mice younger than three days (p = 0.03), confirming glucose-dependency of cellular respiration at the younger ages (Table 1 and Fig. 2).

Table 1
Rates of forebrain cellular respiration (k_c) in phosphate-buffered saline (PBS) supplemented with 10 mM glucose or 0.1 mg/mL glucose oxidase

Age k_c (μM O₂ min^–1 mg^–1) P-value

PBS + glucose PBS + glucose oxidase

≤3 days 0.48 ± 0.11 (22) 0.35 ± 0.08 (15) <0.001

>3 days 0.54 ± 0.14 (13) 0.42 ± 0.09 (13) 0.02

P-value 0.14 0.03

Age	k_c (μM O₂ min^–1 mg^–1)	P-value
≤3 days	0.48 ± 0.11 (22)	0.35 ± 0.08 (15)	<0.001
>3 days	0.54 ± 0.14 (13)	0.42 ± 0.09 (13)	0.02
P-value	0.14	0.03

Values are mean ± SD (n). P value as per the Student t test. The initial rate of respiration (k, in μM O₂ min^–1) was set as the slope of the plot of [O₂] vs. time (linear fit with R² ≥0.970).

Fig.2

Rates of cellular respiration (mean and SD of k_c) in PBS supplemented with 10 mM glucose or 0.1 mg/mL glucose oxidase as function of age. Forebrain cellular respiration (5 to 10 separate experiments per data point) was measured in PBS with 10 mM glucose or PBS with 0.1 glucose oxidase (to deplete residual glucose in the interstitial fluid). The initial rate of respiration (k, in μM O₂ min^–1) was set as the slope of the plot of [O₂] vs. time (linear fit with R ² ≥0.970; see Table 1).

3.4 Cellular ATP

Cellular ATP (both per tissue dry weight and per amount of cellular protein) was higher in pups less than three days of age when compared to older mice (Table 2). The difference between the two groups was significant (p = 0.04) based on nmol cellular ATP per mg tissue dry weight (1.24 ± 0.50 vs 0.75 ± 0.37). The first-order rate constant for forebrain cellular ATP decay in in pups less than three days of age was 44% lower (p = 0.03) than that in older mice (Table 2) and reached its nadir within four minutes (Fig. 3).

Table 2
Forebrain cellular ATP and first-order rate constants of cellular ATP consumption as function of age

Age Cellular ATP (nmol/mg protein)^a First-order rate constants of ATP consumption (min^–1)^b

≤3 days 14.9 ± 8.3 (6) 0.43 ± 0.01

(3) 3 < days≤14 11.2 ± 5.1 (8) 0.77 ± 0.17

(3) P-value 0.32 0.03

Age	Cellular ATP (nmol/mg protein)^a	First-order rate constants of ATP consumption (min^–1)^b
≤3 days	14.9 ± 8.3 (6)	0.43 ± 0.01
(3) 3 < days≤14	11.2 ± 5.1 (8)	0.77 ± 0.17
(3) P-value	0.32	0.03

Values are mean ± SD (n). P value as per the Student t test. ^aForebrain specimens were collected from the studied mice and processed immediately for measuring cellular ATP as described in Methods. ^bForebrain specimens were collected from the studied mice and incubated at 25°C for up to 5 min in PBS with 0.1 mg/mL glucose oxidase. Fragments were removed from the incubation solutions every 60 sec and processed for ATP determination. The first-order rate constants for ATP consumptions were set as the negative of the slopes (R² ≥0.945) of the plots “natural logarithm of the fraction of ATP remaining with time“, as shown in Fig. 2 (three separate experiments per age group).

Fig.3

Rates of cellular ATP consumption (mean and SD) as function of age. Forebrain fragments were collected from the studied mice and incubated at 25°C in PBS with 0.1 mg/mL glucose oxidase (catalyzes the reaction D-glucose + O₂ ⟶ D-glucono-δ-lactone + H₂O₂). Fragments were removed from the incubation solutions every 60 sec and processed for ATP determination. The first-order rate constants of cellular ATP decay (min^–1, Table 2) were set as the negative of the slopes (R ² ≥0.945) of the plots “natural logarithm of the fraction of ATP remaining with time“; three separate experiments per data point.

Cellular ATP in both age groups, both per tissue dry weight and per amount of cellular protein, decreased significantly (p < 0.05) after the addition of glucose oxidase and more so after the addition of cyanide (Table 3). The values for pups less than three days of age were lower than those for older mice (Table 3).

Table 3

Depletion of forebrain cellular ATP following in vitro incubation in PBS with glucose oxidase or glucose oxidase plus cyanide

Age	In vitro incubations^a		P-value
	PBS + glucose oxidase	PBS + glucose oxidase + cyanide
≤3 days	0.7 ± 0.4 (4)	0.1 ± 0.2 (4)	0.06
3<days ≤14	1.1 ± 0.3 (4)	0.2 ± 0.2 (4)	0.002
P-value	0.20	0.40

Values (nmol/mg protein) are mean ± SD (n). P value as per the Student t test. ^aForebrain specimens were collected from the studied mice and incubated at 25°C for 15 min in phosphate-buffered saline (PBS) with 0.1 mg/mL glucose oxidase or PBS supplemented with 0.1 mg/mL glucose oxidase plus 10 mg/mL sodium cyanide. Cellular ATP was then determined at the end of the incubation period.

4 Discussion

The analysis of four age groups between day 0 and day 14 showed that both cellular O₂ consumption and ATP change around the age of three days. In pups younger than three days, both cellular O₂ consumption and ATP content decrease significantly with glucose depletion as compared to older mice (Tables 1 and 2). These findings are consistent with previous studies that have highlighted the vulnerability of the immature brain to decreased availability of glucose and other nutrients [9, 10].

Without a constant flux of glucose to produce ATP via cellular respiration, the brain tissue utilizes first the limited endogenous intracellular fuels with the resulting decrease in the rate of O₂ consumption (Fig. 1A). This reflects sufficient endogenous metabolic fuels (including fatty acid oxidation, which is relatively limited in the neonates) to drive cellular respiration for a few minutes. Endogenous intracellular fuels also include glycogen [17], and become rapidly depleted in the absence of external glucose [18]. Because of the tight coupling between the glycolytic rate and the cellular respiration, in the absence of adequate amount of endogenous substrate glycolysis depends primarily on exogenous glucose. Confirming earlier studies, we found that these endogenous substrates can neither maintain high respiration nor generate ATP in the presence of cyanide that blocks the mitochondrial respiratory chain(Fig. 1B and Table 2) [18].

In younger pups without a constant flux of glucose, the marked decrease in cellular O₂ consumption and ATP content (Table 4) confirms their brain vulnerability to hypoglycemia [9, 10]. The decrease in ATP content indicates that cellular energy is rapidly depleted as a result of fuel unavailability. This is because neuronal cells are highly dependent on oxidative phosphorylation to generate the energy required to fuel their function [16 , 21].

Table 4
Cellular bioenergetics in the very young pups compared with the older ones (set as a reference)

Cellular respiration Cellular ATP content Cellular ATP hydrolysis

PBS + glucose PBS alone PBS + glucose PBS alone

↓ ↓↓ ↔ ↓↓ ↓↓

Cellular respiration	Cellular ATP content	Cellular ATP hydrolysis
↓	↓↓	↔	↓↓	↓↓

The strengths of this study include that, unlike previous experimental studies [21 –25], it has removed any possible confounding by pre-existing diabetes or insulin administration. This study has also limitations. With the experiments focusing only on forebrain tissue, generalization to other areas of the brain cannot be made. The neurons in the hippocampus, the superficial layers of the cortex, the striatum, the occipital and parietal cortex as well as the subcortical white matter are more vulnerable to hypoglycemia [29 –34]. This is perhaps because their mitochondria behave differently with hypoglycemia [23], and also because the lower basal level of antioxidant enzymes in the cortex increases the risk of oxidative damage [35]. This regional susceptibility of the occipital and parietal white matter to hypoglycemic brain injury has also been reported in infants [36 –39]. Another limitation is that, once the brain tissue has been removed from the living organism its environment suddenly changes (cessation of circulation, absence of compensatory or adaptation phenomena). The results of this study may therefore not truly reflect the effects of hypoglycemia in the living organism. Furthermore, as the normal metabolic adaptation to postnatal life also involves the use of lactate [11, 12], and ketone bodies [3] as alternative substrates for oxidative metabolism, the true effects of hypoglycemia in the living organism cannot be answered solely by this study. The friability of the brain tissue is likely to have caused continuing fragmentation during the experiment caused by continuous mixing in the solution, resulting perhaps in a decrease in the accuracy of the reported results of O₂ consumption by tissue weight.

This study was designed to develop and test an experimental model. It included therefore no assessment of the resulting cellular injury or death (caspases), no histopathological correlation and, obviously, no correlation with blood glucose levels. Such studies will now be performed in future experiments.

This experimental model will allow in the future to repeat these experiments as a function of the duration of glucose deprivation and also in different parts of the brain. Measuring the status of the intracellular substrates during glucose deprivation in newborn pups will further clarify their role in such a situation. The experiments will also be performed in-vivo in preterm pups, growth retarded mice, and those with hyperinsulinism caused by induced maternal diabetes. Studying the resulting cellular injury or death (caspases) as well as pathological correlation studies will be also possible. Other in-vivo experiments will include hypoglycemia induced by prolonged fasting or by insulin administration, with correlation of the results with the blood glucose level.

5 Conclusion

As the mechanisms underlying the effects of hypoglycemia on the brain of newborns cannot be directly studied in humans, this animal model offers some answers to clinicians dealing with neonates. Glucose-driven cellular respiration and ATP synthesis are lower in the brain of young pups than that in older mice. In the absence of glucose, endogenous metabolic fuel-driven cellular respiration and ATP synthesis are also lower in the young pups, explaining the special sensitivity of the neonatal brain to hypoglycemia.

Funding

None.

Conflict of interest

The authors declare that they have no conflict of interest.

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Age	k_c (μM O₂ min^–1 mg^–1)		P-value
	PBS + glucose	PBS + glucose oxidase
≤3 days	0.48 ± 0.11 (22)	0.35 ± 0.08 (15)	<0.001
>3 days	0.54 ± 0.14 (13)	0.42 ± 0.09 (13)	0.02
P-value	0.14	0.03

Forebrain cellular bioenergetics in neonatal mice

Abstract

BACKGROUND:

AIMS:

STUDY DESIGN:

RESULTS:

CONCLUSIONS:

Keywords

1 Introduction

2 Methods

2.1 Principles

2.2 Ethical approval

2.3 Animals

2.4 Measurement of O2 consumption

2.5 Measurement of cellular ATP

2.6 Analysis

3 Results

3.1 Descriptive analysis

3.2 Proof of concept validity

Table 4 Cellular bioenergetics in the very young pups compared with the older ones (set as a reference) Cellular respiration Cellular ATP content Cellular ATP hydrolysis PBS + glucose PBS alone PBS + glucose PBS alone ↓ ↓↓ ↔ ↓↓ ↓↓

Funding

Conflict of interest

References

2.4 Measurement of O₂ consumption

Table 4
Cellular bioenergetics in the very young pups compared with the older ones (set as a reference)

Cellular respiration Cellular ATP content Cellular ATP hydrolysis

PBS + glucose PBS alone PBS + glucose PBS alone

↓ ↓↓ ↔ ↓↓ ↓↓