INTRODUCTION

Alzheimer’s disease (AD) is the most common neurodegenerative disorder associated with gradual deterioration in memory and cognitive functions. The disease commonly exists in two forms, familial (fAD) and sporadic (sAD), based upon its origin1. The familial AD, though rare (5%), has been extensively studied due to availability of many transgenic animal models. In contrast, sporadic AD which imparts around 95% of total is not so well investigated. Intracerebroventricular (icv) administration of streptozotocin (STZ) in mouse leads to death of neurons and other cells, and is believed to represent sAD. Several molecular studies have shown the resemblance of (icv-STZ) model to AD patients. However, the impact of icv-STZ administration on neuronal and astroglial function is lacking. The present study assessed the neuronal and astroglial metabolic activity in the cerebral cortex and hippocampus of icv-STZ mouse model of AD by ¹H-[¹³C]-NMR spectroscopy in conjunction with an infusion of [1,6-¹³C₂]Glucose and [2-¹³C]Acetate, respectively²

MATERIALS AND METHODS

All animal experiments were performed under approved protocols by the Institutional Animal Ethics Committee of CCMB. Male C57BL6 (6 month old) mice were divided into two groups: icv-STZ (n=15) and SHAM (n=15). Mice were anaesthetized using ketamine:xylazine mixture (100:10 mg/Kg, ip). The heads were shaved and fixed in the Stoelting stereotax. STZ (dissolved in aCSF:citrate buffer) was administered (5 mg/kg, ICV) in ventricles by infusing 2 ul in 5 min³, and scalp was stitched. Memory and brain energy metabolism of mice were assessed 2 months after STZ administration. Memory was assessed by Novel Object Recognition Test (NORT)⁴. For measurement of neurometabolic activity, animals were kept on fasting for 3 hrs. [1,6-¹³C₂]Glucose or [2-¹³C]Acetate were administered in awake mice trough tail vein for 2 min. Blood was collected from retro orbital sinus followed by fixing the brain metabolites using focused beam microwave irradiation (4kw for 1 s) after 7 minutes of [1,6-¹³C₂]Glucose or 10 min for [2-¹³C]Acetate administration. The cerebral cortex and hippocampus were dissected and stored in liq. N2. Metabolites were extracted from frozen tissue. The concentration and ¹³C labeling of amino acids were measured in ¹H-[¹³C]-NMR spectra recorded at 600 MHz Bruker Avance HD III NMR spectrometer. The cerebral metabolic rates of glucose oxidation (CMR_Glc) by glutamatergic, GABAergic neurons and astroglia were determined from the ¹³C labeling of brain amino acids from [1,6-¹³C₂]glucose and [2-¹³C]acetate. Student T test was carried out to understand the statistical significance of difference of measurement in STZ treated and control mice. All the data are presented as Mean±SEM.

RESULTS

The memory analysis using NORT indicates icv-STZ treated mice (28.5±4.6%) spent significantly (p=0.0001) less time with novel object as compared to SHAM operated controls (68.4±4.4%) suggesting memory impairment in these animals. The levels of Glutamate (12.5±0.1 vs 11.7±0.2 mmol/g, p=0.01), GABA (2.4±0.05 vs 2.3±0.03 mmol/g, p=0.02) and NAA (6.6±0.1 vs 6.1±0.1mmol/g, p=0.002) were decreased in the cerebral cortex of icv-STZ treated mice when compared with (Fig. 1). Similar results were seen in the hippocampus (Fig 1). Additionally, there was an increase in the level of inositol (7.3±0.1 vs 7.8±0.2 mmol/g, p=0.01) in the hippocampus. There was no significant change in 13C labeling of amino acids from [2-13C]acetate in STZ-treated mice suggesting STZ treatment have no impact on astroglial metabolic activity in the cerebral cortex (SHAM 0.1±0.01; icv-STZ 0.1±0.01mmol/g/min, p=.0.51) and hippocampus (SHAM 0.1±0.01; icv-STZ :0.1±0.01 mmol/g, p=.0.54). The concentrations of labeled Glu_C4, GABA_C2, Gln_C4, Asp_C3 and Glu_C3 from [1,6-¹³C₂]Glucose were decreased significantly (p<0.01) in the cerebral cortex as well as in the hippocampus of icv-STZ mice when compared with SHAM controls (Table 1). Consequently, the CMRGlc associated with glutamatergic and GABAergic neurons was reduced (p<0.001) in the cerebral cortex and hippocampus of icv-STZ treated mice (Fig. 2).

DISCUSSION

These data suggest streptozotocin administration in ventricles compromised memory, neurometabolic homeostasis, and synaptic transmission associated with glutamatergic and GABAergic neurons in the cerebral cortex and hippocampus. As these parameters are very well-established markers of AD⁵, a detailed investigation of icv-STZ model will provide a better insight of the mechanism involved in the pathology of sporadic AD. This may be useful for the development of therapeutics for sAD.

Acknowledgements

Authors acknowledge the financial support from CSIR-CCMB to conduct this study.