Changes in inflammatory processes associated with selective vulnerability following mild impairment of oxidative metabolism

Abstract

Abnormalities in oxidative metabolism and reductions of thiamine-dependent enzymes accompany many age-related neurodegenerative diseases. Thiamine deficiency (TD) produces a cascade of events including mild impairment of oxidative metabolism, activation of microglia, astrocytes and endothelial cells that leads to neuronal loss in select brain regions. The earliest changes occur in a small, well-defined brain region, the submedial thalamic nucleus (SmTN). In the present study, a micropunch technique was used to evaluate quantitatively the selective regional changes in mRNA and protein levels. To test whether this method can distinguish between changes in vulnerable and non-vulnerable regions, markers for neuronal loss (NeuN) and endothelial cells (eNOS) and inflammation (IL-1beta, IL-6 and TNF-alpha) in SmTN and cortex of control and TD mice were assessed. TD significantly reduced NeuN and increased CD11b, GFAP and ICAM-1 immunoreactivity in SmTN as revealed by immunocytochemistry. When assessed on samples obtained by the micropunch method, NeuN protein declined (-49%), while increased mRNA levels were observed for eNOS (3.7-fold), IL-1beta (43-fold), IL-6 (44-fold) and TNF-alpha (64-fold) in SmTN with TD. The only TD-induced change that occurred in cortex with TD was an increase in TNF-alpha (22-fold) mRNA levels. Immunocytochemical analysis revealed that IL-1beta, IL-6 and TNF-alpha protein levels increased in TD brains and colocalized with glial markers. The consistency of these quantitative results with immunocytochemical measurements validates the micropunch technique. The results demonstrate that TD induces quantitative, distinct inflammatory responses and oxidative stress in vulnerable and non-vulnerable regions that may underlie selective vulnerability.

Fig. 1. TD alters the response of multiple cell types

Adult male C57BL/6 mice were treated with saline or pyrithiamine hydrobromide (0.5 mg/kg; i.p.) once daily for 10 days. Animals were sacrificed on the 10th day and the brains were fixed and processed for NeuN, CD11b, GFAP or ICAM-1 immunostaining. Left and right panels show the control and TD, respectively. NeuN (a, b), CD11b (c, d), GFAP (e, f), ICAM-1 (g, h). Insets show the magnified images of the multiple cell types (n=5). Scale bars: a-h, 200 μm; Insets; a-h, 10 μm.

Fig. 2. Micropunch technique allows sampling of SmTN

The left and right panels show the SmTN and cortex, respectively. Fig. a and b show the location of the SmTN and cortex in the atlas. The arrow in Fig. c and d points to the NeuN immunoreactivity in control SmTN and cortex respectively. The arrow in Fig. e and f reveals the loss of NeuN immunoreactivity in TD SmTN but not in cortex. Fig. g and h show the section after it has been micropunched using the Harris Uni-Core™, Size 0.75mm. Scale bars: c-h, 1 mm.

Fig. 3. TD decreased NeuN protein levels

Western blots were performed with the protein homogenates prepared from the SmTN and cortex micropunches of three control and TD brains probed with NeuN antibody and visualized by LI-COR IR dyes (A). Panel (B) shows the densitometric analysis of band intensity for NeuN (48 and 46 kDa) were quantified using Odyssey software. Data are represented as mean ± SEM in each group. * p <0.05 compared with control group (n=2). Although the statistics were done with n’s of two, a total of six animals in each group were used. Qualitatively similar results were observed with two more control and two more TD samples when analysis was done by ECL.

Fig. 4. TD increased mRNA and protein levels of endothelial NOS

Quantitative RT-PCR of eNOS mRNA was performed in total RNA extracts from SmTN and cortex punches of three control or TD brains. The expression levels of mRNA were normalized relative to the levels of GAPDH mRNA and fold differences were calculated relative to the control group. Values are mean ± SEM in each group. * p <0.05 compared with control group (n=3) (A). Values are mean ± SEM in each group. * p <0.05 compared with control group (n=3). Although the statistics were done with n’s of three, the results reflect a total of nine animals in each group. Panel (B) shows the representative control and TD brains sections stained with an eNOS antibody. TD-10 brains showed increased eNOS immunoreactivity (n=4). Scale bars: 100 μm; Insets 20 μm.

Fig. 5. TD increased mRNA and protein levels of pro-inflammatory markers

Quantitative RT-PCR of pro-inflammatory markers IL-1β, IL-6 and TNF-α mRNA was performed on total RNA extracts from SmTN and cortex punches of three control or TD 10 brains. The expression levels of mRNA were normalized relative to the levels of GAPDH mRNA and fold differences were calculated relative to the control group. Values are mean ± SEM in each group. * p <0.05 compared with control group (n=3). Although the statistics were done with n’s of three, the results reflect a total of nine animals in each group. Panel (B) shows the representative TD-10 brains sections stained with IL-1β, IL-6 and TNF-α in SmTN region and colocalization with glial markers. IL-1β and TNF-α (green) double immunofluoresence with CD11b (red) showed that IL-1β and TNF-α colocalized in microglia (yellow). Reactive astrocytes labeled with GFAP (red) showed IL-6 (green) colocalized with GFAP (yellow) (n=4). Scale bars: 100 μm.

Fig. 5. TD increased mRNA and protein levels of pro-inflammatory markers

Quantitative RT-PCR of pro-inflammatory markers IL-1β, IL-6 and TNF-α mRNA was performed on total RNA extracts from SmTN and cortex punches of three control or TD 10 brains. The expression levels of mRNA were normalized relative to the levels of GAPDH mRNA and fold differences were calculated relative to the control group. Values are mean ± SEM in each group. * p <0.05 compared with control group (n=3). Although the statistics were done with n’s of three, the results reflect a total of nine animals in each group. Panel (B) shows the representative TD-10 brains sections stained with IL-1β, IL-6 and TNF-α in SmTN region and colocalization with glial markers. IL-1β and TNF-α (green) double immunofluoresence with CD11b (red) showed that IL-1β and TNF-α colocalized in microglia (yellow). Reactive astrocytes labeled with GFAP (red) showed IL-6 (green) colocalized with GFAP (yellow) (n=4). Scale bars: 100 μm.