Astrocytes as a Predominant Cellular Site of ^99mTc-HMPAO Retention

Abstract

Technetium-99m-d,l-hexamethylpropylene amine oxime ((99m)Tc-HMPAO) retention in the brain monitored by single photon emission computed tomography (SPECT) is currently used as a marker of cerebral blood flow. The purported mechanism by which (99m)Tc-HMPAO accumulates in the brain is through its intracellular conversion from a lipophilic form to more hydrophilic derivatives within the brain parenchyma. The issue of the contribution of different cell types on (99m)Tc-HMPAO retention was investigated in vitro by studying the accumulation of (99m)Tc-HMPAO in primary cultures of mouse cortical astrocytes and neurons. Results show that (99m)Tc-HMPAO retention predominates in astrocytes over neurons by a factor of approximately 2.5 (0.26 +/- 0.05 vs. 0.095 +/- 0.042 fmol/mg protein after 120 minutes, respectively). Diethyl maleate (60 micromol/L), ethacrynic acid (1 mmol/L) and buthionine sulfoximine (1 mmol/L), 3 agents which significantly reduced glutathione levels also decreased (99m)Tc-HMPAO retention in both astrocytes (29%, 3%, and 46% of control, respectively) and neurons (69%, 11% and 63% of control). Decrease did not always correlate with glutathione levels, however, which suggests that other factors could be involved. The possibility that cell energy status determines (99m)Tc-HMPAO retention was also assessed. Agents that activate (glutamate, azide) or inhibit (cytochalasin B) glucose utilization in astrocytes, as measured by the (3)H-2-deoxyglucose method, were without effect on (99m)Tc-HMPAO retention. In conclusion, the data presented indicate that astrocytes may constitute a prominent site of (99m)Tc-HMPAO retention and most likely contribute significantly to the SPECT signal. In addition, the data also suggest that specific alterations in glial cell metabolism could explain flow-independent changes in (99m)Tc-HMPAO retention in the brain as observed by SPECT in some pathologies.

Modulation of astrocytic metabolic phenotype by proinflammatory cytokines

“…Reasons for such discrepancies are not known but may arise from different culture conditions and in particular composition of media that are used. Nevertheless, these values are in good agreement with those measured in a study using the same culture paradigm (Zerarka et al, 2001). A striking feature of glutathione metabolism in astrocytes is the release of large amounts of glutathione in the extracellular space (Dringen, 2000).…”

“…Glutathione levels (reduced form, GSH or oxidized form, GSSG) were determined as previously described (Zerarka et al, 2001). For these experiments, 24 h before treatment cell culture medium was replaced by a phenol red free culture medium (D2902, Sigma-Aldrich) supplemented with 44 mM NaHCO 3 , 10 mL/L of an antibiotic/ antimycotic solution (A7292, Sigma-Aldrich) and 10% FCS and complemented to 25 mM glucose to give rise to a medium that is strictly equivalent to the normal culture medium except for phenol red.…”

Endothelial Cell-Derived Nitric Oxide Enhances Aerobic Glycolysis in Astrocytes via HIF-1 -Mediated Target Gene Activation

“…Primary cortical neuronal cultures were obtained from mice at embryonic day 18 as described previously (Zerarka et al, 2001). In brief, cortices were isolated and neurons mechanically dissociated.…”

Overview of Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT) Methodologies

“…Since its extraction into brain and subsequent retention is so extensive, a SPECT recording made some minutes after inhalation shows the pattern of CBF. However, as noted above, [ 99m Tc]-HMPAO entrapment in brain is complex; Studies in vitro indicate that the preponderant cellular site of trapping is not in neurons, but in glia, in a manner related to the presence of glutathione (Zerarka et al 2001 ) ; long term retention of the tracer in brain thus involves metabolic conversion of the tracer into a hydrophilic metabolite, with more constrained diffusion across plasma membranes. As such, […”