Protein-Energy Malnutrition Developing after Global Brain Ischemia Induces an Atypical Acute-Phase Response and Hinders Expression of GAP-43

<div><p>Protein-energy malnutrition (PEM) is a common post-stroke problem. PEM can independently induce a systemic acute-phase response, and pre-existing malnutrition can exacerbate neuroinflammation induced by brain ischemia. In contrast, the effects of PEM developing in the post-ischemic period have not been studied. Since excessive inflammation can impede brain remodeling, we investigated the effects of post-ischemic malnutrition on neuroinflammation, the acute-phase reaction, and neuroplasticity-related proteins. Male, Sprague-Dawley rats were exposed to global forebrain ischemia using the 2-vessel occlusion model or sham surgery. The sham rats were assigned to control diet (18% protein) on day 3 after surgery, whereas the rats exposed to global ischemia were assigned to either control diet or a low protein (PEM, 2% protein) diet. Post-ischemic PEM decreased growth associated protein-43, synaptophysin and synaptosomal-associated protein-25 immunofluorescence within the hippocampal CA3 mossy fiber terminals on day 21, whereas the glial response in the hippocampal CA1 and CA3 subregions was unaltered by PEM. No systemic acute-phase reaction attributable to global ischemia was detected in control diet-fed rats, as reflected by serum concentrations of alpha-2-macroglobulin, alpha-1-acid glycoprotein, haptoglobin, and albumin. Acute exposure to the PEM regimen after global brain ischemia caused an atypical acute-phase response. PEM decreased the serum concentrations of albumin and haptoglobin on day 5, with the decreases sustained to day 21. Serum alpha-2-macroglobulin concentrations were significantly higher in malnourished rats on day 21. This provides the first direct evidence that PEM developing after brain ischemia exerts wide-ranging effects on mechanisms important to stroke recovery.</p></div

Representative photographs of the Iba-1 (A) and GFAP (B) immunofluorescence in the CA1 hippocampal subregion on day 21 after global brain ischemia.

<p>Blue  =  DAPI (cell nuclei). Red  =  Iba-1 (activated microglia marker; A) or GFAP (astrocytic marker; B).</p

Representative photographs of GAP-43 (A), synaptophysin (B) and SNAP-25 (C) immunofluorescence in the CA3 mossy fibers on day 21 after global brain ischemia.

<p>Blue  =  DAPI (cell nuclei). Green  =  GAP-43 (A), synaptophysin (B), or SNAP-25 (C).</p

PEM introduced on day 3 after global ischemia did not alter the hippocampal glial response on either day 5 or 21.

<p>Immuno-labeling results are shown for Iba-1 in the CA1 (A) and CA3 (B) and for GFAP in the CA1 (C) and CA3 (D) hippocampal subregions.*Indicates a significant difference for the <i>posthoc</i> comparisons made by Tukey's Test (CON-Sham versus CON-ISC) within the specific time-point (p<0.05). Results are shown as mean ± SEM integrated density value (IDV).</p

PEM initiated at 3 days after global brain ischemia did not exacerbate hippocampal CA1 neuronal death.

<p>Data are presented as mean ± SEM. *CA1 neuronal counts were significantly decreased in the CON-ISC group, as compared to the CON-Sham group by 1- factor ANOVA and Tukey's test (p<0.001), but the CON-ISC and PEM-ISC groups did not differ (p>0.87).</p><p>PEM initiated at 3 days after global brain ischemia did not exacerbate hippocampal CA1 neuronal death.</p

The influence of post-ischemic PEM on expression of GAP-43 (A), synaptophysin (B) and SNAP-25 (C) within the CA3 mossy fibers on days 5 and 21 following global brain ischemia.

<p>*Indicates a significant difference for either of the 2 <i>posthoc</i> comparisons made by Tukey's Test (CON-Sham versus CON-ISC and CON-ISC versus PEM-ISC) within the specific time-point (p<0.05). α Indicates a significant difference between CON-ISC and PEM-ISC groups detected by unadjusted pairwise comparison (p<0.05). Results are shown as mean ± SEM integrated density value (IDV).</p

The PEM regimen introduced on day 3 after global brain ischemia depressed body weight and food intake.

<p>Data are shown as mean ± SEM for the day 21 treatment groups. The dashed vertical line illustrates the day on which rats were assigned to experimental diet. (<b>A</b>) Body weights are shown for days 3, 7, 14 and 21 (CON-Sham21d, n = 8; CON-ISC21d, n = 11; PEM-ISC21d, n = 11).*Indicates a significant effect of experimental diet on body weight (PEM-ISC compared to CON-Sham and CON-ISC groups) by Tukey's Test (p<0.05). (<b>B</b>) Food intake was collected daily on a cage basis (CON-Sham21d + CON-ISC21d, n = 8 cages [2–3 rats/cage]; PEM-ISC21d, n = 5 cages [2–3 rats/cage]) and calculated as daily cage food intake/number of rats per cage. γ Indicates the first day on which PEM-ISC rats experienced a significant reduction in food intake, when compared to that for the combined CON groups, as detected by an independent-sample Student's t-test (p<0.05).</p

PEM introduced on day 3 following global brain ischemia elicits an atypical acute-phase response.

<p>Alpha-2-macroglobulin (A2M) (<b>A</b>), alpha-1-acid glycoprotein (AGP) (<b>B</b>), and haptoglobin (<b>C</b>) are positive acute-phase proteins, and albumin (<b>D</b>) is a negative acute-phase protein. *Indicates a significant difference for the 2 <i>posthoc</i> comparison made by Tukey's Test (CON-ISC versus PEM-ISC) within the specific time-point (p<0.05). Data are shown as mean ± SEM for each experimental group on day 5 (CON-Sham, n = 8; CON-ISC, n = 11; PEM-ISC, n = 6) and day 21 (CON-Sham, n = 8; CON-ISC, n = 6; PEM-ISC, n = 10).</p

Subcellular Biochemical Investigation of Purkinje Neurons Using Synchrotron Radiation Fourier Transform Infrared Spectroscopic Imaging with a Focal Plane Array Detector

Coupling Fourier transform infrared spectroscopy with focal plane array detectors at synchrotron radiation sources (SR-FTIR-FPA) has provided a rapid method to simultaneously image numerous biochemical markers in situ at diffraction limited resolution. Since cells and nuclei are well resolved at this spatial resolution, a direct comparison can be made between FTIR functional group images and the histology of the same section. To allow histological analysis of the same section analyzed with infrared imaging, unfixed air-dried tissue sections are typically fixed (after infrared spectroscopic analysis is completed) via immersion fixation. This post fixation process is essential to allow histological staining of the tissue section. Although immersion fixation is a common practice in this filed, the initial rehydration of the dehydrated unfixed tissue can result in distortion of subcellular morphology and confound correlation between infrared images and histology. In this study, vapor fixation, a common choice in other research fields where postfixation of unfixed tissue sections is required, was employed in place of immersion fixation post spectroscopic analysis. This method provided more accurate histology with reduced distortions as the dehydrated tissue section is fixed in vapor rather than during rehydration in an aqueous fixation medium. With this approach, accurate correlation between infrared images and histology of the same section revealed that Purkinje neurons in the cerebellum are rich in cytosolic proteins and not depleted as once thought. In addition, we provide the first direct evidence of intracellular lactate within Purkinje neurons. This highlights the significant potential for future applications of SR-FTIR-FPA imaging to investigate cellular lactate under conditions of altered metabolic demand such as increased brain activity and hypoxia or ischemia