In situ biospectroscopic investigation of rapid ischemic and postmortem induced biochemical alterations in the rat brain

© 2014 American Chemical Society. Rapid advances in imaging technologies have pushed novel spectroscopic modalities such as Fourier transform infrared spectroscopy (FTIR) and X-ray absorption spectroscopy (XAS) at the sulfur K-edge to the forefront of direct in situ investigation of brain biochemistry. However, few studies have examined the extent to which sample preparation artifacts confound results. Previous investigations using traditional analyses, such as tissue dissection, homogenization, and biochemical assay, conducted extensive research to identify biochemical alterations that occur ex vivo during sample preparation. In particular, altered metabolism and oxidative stress may be caused by animal death. These processes were a concern for studies using biochemical assays, and protocols were developed to minimize their occurrence. In this investigation, a similar approach was taken to identify the biochemical alterations that are detectable by two in situ spectroscopic methods (FTIR, XAS) that occur as a consequence of ischemic conditions created during humane animal killing. FTIR and XAS are well suited to study markers of altered metabolism such as lactate and creatine (FTIR) and markers of oxidative stress such as aggregated proteins (FTIR) and altered thiol redox (XAS). The results are in accordance with previous investigations using biochemical assays and demonstrate that the time between animal death and tissue dissection results in ischemic conditions that alter brain metabolism and initiate oxidative stress. Therefore, future in situ biospectroscopic investigations utilizing FTIR and XAS must take into consideration that brain tissue dissected from a healthy animal does not truly reflect the in vivo condition, but rather reflects a state of mild ischemia. If studies require the levels of metabolites (lactate, creatine) and markers of oxidative stress (thiol redox) to be preserved as close as possible to the in vivo condition, then rapid freezing of brain tissue via decapitation into liquid nitrogen, followed by chiseling the brain out at dry ice temperatures is required

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

Preventing protein-energy malnutrition after cortical stroke enhances recovery of symmetry in forelimb use during spontaneous exploration

Protein-energy malnutrition (PEM) commonly arises after stroke. We investigated the effects of preventing PEM on spontaneous recovery of forelimb use, infarct size, and the acute phase response in the chronic post-stroke period. Male, adult, Sprague–Dawley rats were acclimatized to control diet (12.5% protein), tested for pre-stroke forelimb use symmetry in the cylinder test, and exposed to photothrombotic cortical stroke or sham surgery. Food intake was monitored daily, and body weight weekly. Forelimb use was tested on day 4 after surgery, before assignment to control diet or PEM (0.5% protein), with subsequent testing on days 16 and 29. Blood, brain, and liver were collected on day 30. The low protein diet resulted in PEM, measured by decreased body weight (p < 0.001) and food intake (p = 0.016) and increased liver lipid (p < 0.001). Stroke (p = 0.016) and PEM (p = 0.001) independently elicited increases in serum α-2-macroglobulin concentration, whereas PEM alone decreased albumin (p < 0.001). PEM reduced recovery of forelimb use symmetry during exploration on days 16 (p = 0.024) and 29 (p = 0.013) but did not influence infarct size (p = 0.775). Stroke reduced reliance on the stroke-affected forelimb to initiate exploration up until day 29 (p < 0.001); PEM had no influence (p ≥ 0.463). Preventing post-stroke PEM appears to yield direct benefits for certain types of motor recovery.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

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

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

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