PURPOSE

Hyperglycemia (blood glucose >150 mg/dL) is common in extremely low-gestational-age neonates (ELGAN, gestational age 24 - 28 week at birth) with an incidence of 40 - 80% ^1,2. Relative hypoinsulinism combined with the need for dextrose infusion for nutrition are responsible for hyperglycemia in these infants. Hyperglycemia increases the risk of death, intraventricular hemorrhage and sepsis in the neonatal period ³. The long-term effects are not well understood. Limited data demonstrate that recurrent neonatal hyperglycemia is associated with deceleration of somatic and brain growth that lasts at least until the age of two years in ELGANs ^2,4 and with neurodevelopmental deficits at two years of age ⁵. The adverse effects were directly proportional to the severity of hyperglycemia in the neonatal period ^2,4. In other conditions (for example, diabetes mellitus), hyperglycemia causes structural and functional changes in the hippocampus ^6,7. Long-term structural and functional hippocampus-specific deficits are common in ELGANs ⁸. It is not known whether hyperglycemia has a role in these deficits. The purpose of the study was to determine whether recurrent hyperglycemia in the neonatal period leads to long-term alterations in the neurochemical profile of the hippocampus using a rat model of neonatal hyperglycemia.

METHODS

Sprague-Dawley rat pups were subjected to hyperglycemia of 2-hour duration, twice daily from postnatal day 3 (P3, stage of brain development = human ELGAN) to P12 (stage of brain development = human full term neonate) using subcutaneous injection of 30% dextrose (moderate hyperglycemia - MODERATE-HG) group or 50% dextrose (severe hyperglycemia - SEVERE-HG) group. Littermates in the control group (CONTROL) were injected with normal saline. The neurochemical profiles of the hippocampus were determined in the CONTROL (N = 6), MODERATE-HG (N = 6) and SEVERE-HG (N = 7) group on P30 (stage of brain development = 2-year-old human child) using ¹H MRS at 9.4T. Data were acquired using FASTMAP shimming ⁹ and ultra-short TE STEAM (TE = 2 ms) localization sequence combined with VAPOR water suppression ¹⁰. Metabolites were quantified using LCModel with the spectrum of fast relaxing macromolecules included in the basis set. Spontaneously breathing animals were anesthetized with 1.0 – 1.5% isoflurane. Brain tissue was harvested and processed for histochemical assessment of synaptic structure (b-tubulin histochemistry) and astrocytosis (S100b histochemistry). The intergroup differences in metabolite concentrations and histochemistry were determined using ANOVA and t-tests.

RESULTS

The blood glucose concentration (mg/dL) was 139 ± 3, 226 ± 12 and 380 ± 27 in the CONTROL group, MODERATE-HG group and SEVERE-HG group, respectively (p < 0.01). The spectral quality consistently accomplished in this study (Fig. 1) enabled reliable quantification of seventeen brain metabolites (Fig. 2). Small but significant changes (p < 0.05) were observed for lactate concentration as well as for glutamate/glutamine and phosphocreatine/creatine ratios in hyperglycemia groups relative to controls. The Lac levels and Glu/Gln ratios progressively decreased with the severity of the neonatal hyperglycemia. The PCr/Cr ratio increased in hyperglycemia groups relative to controls. Histochemistry demonstrated decreased dendritic arborization density (lower β-tubulin density) and increased astrocytosis (higher number of S100b-positive cells) in the hippocampus of the MODERATE-HG and SEVERE-HG groups.

DISCUSSION

Recurrent neonatal hyperglycemia altered the neurochemical profile and structure of the developing rat hippocampus. The presence of changes at P30, despite cessation of hyperglycemia on P12, indicates that neonatal hyperglycemia may cause long-term effects in the developing brain. Observed changes in metabolites directly involved in brain energy metabolism (PCr/Cr, Lac) indicate decreased hippocampal activity ¹¹. Moreover, decreased Glu/Gln ratio implies altered glutamate-glutamine cycling between neurons and glia ¹¹. Alterations in energy metabolism and glutamate-glutamine cycling are in a good agreement with the decreased synaptic density observed in the hyperglycemia groups. Astrocytosis may also contribute to the lower Glu/Gln ratio in the hyperglycemia group, since Glu and Gln are primarily localized in neuronal and astrocytic compartments, respectively. A similar pattern of neurochemical changes has been linked to cognitive impairments in adults with Alzheimer disease ^12,13.

CONCLUSIONS

The results of this study indicate that the recurrent hyperglycemia during neonatal period may alter energy metabolism and glutamatergic neurotransmission, which can contribute to delayed hippocampal development and cognitive deficits in ELGANs.

Acknowledgements

Supported by: NIH grants P41 EB015894, P30 NS076408 and WM KECK Foundation

References

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