001 - Glutamine and Hypoxic Brain Injury: A Mechanistic Insight.

Publication Number: 1.5409

Marissa G. Burd, St. Christopher's Hospital for Children, Philadelphia, PA, United States; John R. Grothusen, Drexel University, Haddon Townshop, NJ, United States; Ferit Tuzer, Drexel University, Philadelphia, PA, United States; Timothy Elton, St. Christopher's Hospital for Children, Pennsauken, NJ, United States; Shadi N. Malaeb, Drexel University College of Medicine, Philadelphia, PA, United States

Background: Despite current therapies, more than 40% of infants with hypoxic ischemic encephalopathy have long-term neurodevelopmental impairment. The mechanisms of hypoxic brain injury (HBI) affect multiple cell lines, including astrocytes. Under normal conditions, astrocytes convert glutamate (GLU) and ammonia into glutamine (GLN) via glutamine synthase. However, under hypoxic conditions, astrocytes undergo activation, and adapt their metabolism to help neurons survive. Understanding the mechanism behind astrocyte activation could advance treatment options.

Objective: (1) Determine if GLN and GLU levels in the brain are altered after HBI with and without hypothermia (HT). (2) Evaluate GLN’s effect on astrocyte activation using cell cultures.

Design/Methods: Anesthetized and ventilated piglets (3-5 days old; n = 24) were exposed to either normoxia (Nx) or hypoxia (Hx; FiO2=0.06 x1hr) without reoxygenation, or Hx for 1 hour with reoxygenation for 4 hours at normothermic (HxNT) or hypothermic conditions (HxHT; 33.0±0.5°C), then euthanized for tissue harvest. High performance liquid chromatography was used to determine GLN and GLU levels in the cerebral cortex (CC) for each group. Commercially available human astrocyte cell line cultures were incubated for 24 hours in astrocyte growth media, media with 200mM GLN, or media with 200mM GLN and 3mM glycerol phenylbutyrate (GBP; GLN chelator). Astrocyte morphology was examined microscopically by two observers.

Results: Hypoxia resulted in significant metabolic acidosis, cerebral lactic acidosis, and cerebral edema (Table 1). Hypoxic piglets reoxygenated for 4 hours with or without HT had increased GLN, but decreased GLU in their CC compared to controls (Figure 1). Astrocytes incubated in GLN had an increased number of cellular processes indicative of activated astrocyte morphology. This effect was blunted by a small amount of GLN chelator (Figure 2).

Conclusion(s): We observed increased glutamine and decreased glutamate in the piglet brain after hypoxia regardless of hypothermia and that glutamine induced astrocyte activation in cell culture. We suspect hypoxia-induced cytotoxic edema disrupts cellular morphology and astrocyte-neuronal GLN-GLU shuttles, leading to GLN buildup in the astrocyte, releasing ammonia and inducing reactive astrogliosis. Glycerol phenylbutyrate, a lipophilic triglyceride compound which clears ammonia and is used clinically for urea cycle disorders, blunted the effect of excess GLN on astrocytes. We speculate redirected use of inborn errors of metabolism agents may provide a novel approach to hypoxic brain injury as a metabolic disorder.

Physiologic Changes
Table 1. Arterial pH, base deficit, cerebral lactate levels, and cerebral water content in each of the study groups. * p < 0.05 versus normoxia control; † p < 0.05 versus normal control;
ª p < 0.05 versus hypoxia non-reoxygenated (One-way ANOVA testing). Mean ± SD.

Glutamine and Glutamate Levels in the Cerebral Cortex of Piglets
Figure 1. Levels of glutamine (A) and glutamate (B) in whole tissue extracts from the cerebral cortex of normal, non-instrumented, non-anesthetized piglets (open bars; n=6) and anesthetized sham-control piglets, ventilated for 5 hours with normal oxygen levels (Nx; shaded bars; n=6), or made hypoxic x1hr and either not reoxygenated (Hx, black bars; n=6), or reoxygenated x4hrs in normothermic conditions (HxNT; downward stripes bars; n=7) or under hypothermic conditions (HxHT; 33.0±0.5°C; upward stripes bars; n=5), then euthanized for tissue harvest. One-way ANOVA with post hoc testing was done and found statistically significant increases in the glutamine levels after hypoxia and reoxygenation with and without hypothermic conditions (* p < 0.05 vs. normal; † p < 0.05 vs. normoxia) and statistically significant decreases in the glutamate levels after hypoxia with and without reoxygenation or hypothermia (* p < 0.05 vs. normal; † p < 0.05 vs. normoxia).

Astrocyte Counts in Different Types of Media
Figure 2. Human astrocyte cell line cultures were incubated for 24 hours in triplicate wells containing astrocyte growth media (A), media with 200mM glutamine (B), or media with 200mM of glutamine and 3mM of glycerol phenylbutyrate (C; glutamine chelator). The cell cultures were then examined under light microscopy by two independent observers, and the numbers of cellular processes for each visible astrocyte were counted (95% concordance). Chi-squared statistical analysis was performed. The astrocytes incubated in media with 200mM glutamine showed evidence of activation, with a significant increase in the number of cellular processes (* p < 0.05 vs. astrocyte media). The astrocytes incubated in media with 200mM glutamine and 3mM glycerol phenylbutyrate did not show signs of glutamine activation, with a significantly lower number of cellular processes († p < 0.02 vs. glutamine).