Up-regulation of PK11195 binding in areas of axonal degeneration coincides with early microglial activation in mouse brain.

Increased binding of the peripheral benzodiazepine binding site (PBBS) ligand [(3)H]PK11195 in the central nervous system of patients suffering from acute and chronic neuropathology has been associated with reactive microgliosis. However, it remains uncertain which stages of microglial activation occur in conjunction with the increased [(3)H]PK11195 binding. We used quantitative autoradiography for [(3)H]PK11195 and quantitative polymerase chain reaction for PBBS mRNA and markers of early and late microglial activation to investigate the time-course of cellular responses in the hippocampus of mice with degeneration of the entorhinal-hippocampal perforant path. The axonal lesion evoked an increase in the B(max) for [(3)H]PK11195 in hippocampus which peaked at 2 days post-lesion, remained elevated at day 5 and began to decline at 10 days post-lesion. These changes occurred in the absence of significant changes in affinity in vitro. Quantitative polymerase chain reaction analysis of isolated hippocampi using exon-specific primers indicated the presence of several splice variants of PBBS mRNA, which appeared to be affected differentially by the lesion. The changes in PBBS mRNA and CD11b mRNA levels correlated with the B(max) for [(3)H]PK11195 during 10 days post-lesion, suggesting that microglial activation couples with increases in mRNA levels for these markers. In addition, the onset of changes in PBBS mRNA levels coincided with the significantly elevated tumor necrosis factor mRNA levels present during early microglial activation at 2 days post-lesion. We conclude that up-regulation of [(3)H]PK11195 binding and PBBS mRNA levels coincided with early microglial activation, characterized by concomitantly increased microglial tumor necrosis factor mRNA levels, and persisted throughout the period with reactive microgliosis.

Validation of two reference genes for mRNA level studies of murine disease models in neurobiology.

Reverse transcription of extracted cellular RNA combined with real-time PCR is now an established method for sensitive detection and quantification of specific mRNA level changes in experimental models of neurological diseases. To neutralize the impact of experimental error and make quantification more precise, normalization of test gene data using data from a constantly expressed gene, a reference gene that is tested along with the test gene, is required. There is no single gene constantly expressed under all experimental conditions. For a given set of conditions or a given disease model, identification of an unaffected reference gene is necessary. In this report, we present our findings from evaluation and validation of the genes encoding hypoxanthine guanine phosphoribosyl transferase 1 (HPRT1) and glyceraldehyde phosphate dehydrogenase (GAPDH) as individual reference genes in mRNA level studies involving four murine neurological disease models. We find both genes are suitable as a reference gene with these four models, provided quantification of subtle changes are avoided. We furthermore demonstrate that above a certain threshold of test mRNA level changes and given high quality RNA processing, normalization to total RNA alone provides for equally reliable quantitative mRNA level results.

Distribution of PK11195 binding sites in porcine brain studied by autoradiography in vitro and by positron emission tomography.

The cerebral distribution of peripheral-type benzodiazepine binding sites (PBBS) in human brain has been investigated by positron emission tomography (PET) with the specific radioligand [11C]PK11195 in diverse neuropathological conditions. However, little is known about the pattern of PK11195 binding sites in healthy brain. Therefore, we used quantitative autoradiography to measure the saturation binding parameters for [3H]PK11195 in cryostat sections from young Landrace pigs. Specific binding was lowest in the cerebellar white matter (85 fmol mg(-1)) and highest in the caudate nucleus (370 fmol mg(-1)), superior colliculus (400 fmol mg(-1)), and anterior thalamic nucleus (588 fmol mg(-1)). The apparent affinity was in the range of 2-6 nM in vitro, predicting high specific binding in PET studies of living brain. However, the distribution volume (V(d), ml g(-1)) of high specific activity [11C]PK11195 was nearly homogeneous (3 ml g(-1)) throughout brain of healthy Landrace pigs, and was nearly identical in studies with lower specific activity, suggesting that factors in vivo disfavor the detection of PBBS in Landrace pigs with this radioligand. In young, adult Göttingen minipig brain, the magnitude of V(d) for [11C]PK11195 was in the range 5-10 ml g(-1), and had a heterogeneous distribution resembling the in vitro findings in Landrace pigs. There was a trend toward globally increased V(d) in a group of minipigs with acute MPTP-induced parkinsonism, but no increase in V(d) was evident in the same pigs rescanned at 2 weeks after grafting of fetal mesencephalon to the partially denervated striatum. Thus, [11C]PK11195 binding was not highly sensitive to constituitively expressed PBBS in brain of young Landrace pigs, and did not clearly demonstrate the expected microglial activation in the MPTP/xenograft model of minipigs.