Research ArticleNEUROSCIENCE

Mechanisms of murine cerebral malaria: Multimodal imaging of altered cerebral metabolism and protein oxidation at hemorrhage sites

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Science Advances  18 Dec 2015:
Vol. 1, no. 11, e1500911
DOI: 10.1126/sciadv.1500911
  • Fig. 1 Immunohistochemistry shows compromised BBB and increased fibrinogen content in the brain parenchyma of CM mice.

    (A and B) Immunohistochemical localization of the plasma protein fibrinogen in the cerebellum of control (A) and CM (B) mice. Brown DAB staining, indicating increased BBB compromise and leakage of the plasma protein fibrinogen into the brain parenchyma, was observed in mice with CM. Counterstaining with hematoxylin revealed numerous leukocyte nuclei within the microvessels (arrows) in CM mice. WM, white matter; GM, gray matter.

  • Fig. 2 Multimodal biospectroscopic approach to studying the biochemical mechanisms of CM.

    (A to G) Biospectroscopic imaging in combination with histology of cerebellum tissue in CM and control mice. (A) Representative example of H&E histology of cerebellum tissue from control (i) and CM (ii) mice, highlighting the appearance of tissue edema at the site of vascular hemorrhage. (B) XFM elemental mapping of the distribution of Fe in control (i) and surrounding hemorrhaged tissue in CM (ii) cerebellum tissue. (C) Resonance Raman mapping of the distribution of hemoglobin in control (i) and hemorrhaged tissue in CM (ii) cerebellum tissue. (D) FTIR imaging of the lipid (second-derivative band intensity at 1742 cm−1) distribution in control (i) and hemorrhaged tissue in CM (ii) cerebellum tissue. (E) FTIR imaging of the lactate (second-derivative band intensity at 1127 cm−1) distribution in control (i) and hemorrhaged tissue in CM (ii) cerebellum tissue. (F) FTIR imaging of the α helix protein (second-derivative band intensity at 1656 cm−1) distribution in control (i) and hemorrhaged tissue in CM (ii) cerebellum tissue. (G) FTIR imaging of the aggregated β sheet protein (second-derivative band intensity at 1627 cm−1) distribution in control (i) and hemorrhaged tissue in CM (ii) cerebellum tissue. Black arrows in (Fii) and (Gii) highlight the location of elevated aggregated β sheet/α helix protein ratio hotspots. Scale bar in (A), 50 μm. (H to K) Representative second derivatives of the average FTIR spectra from control and CM mouse cerebellum tissue. (H) Lipid and protein alterations in the spectra from hemorrhaged and nonhemorrhaged inner white matter from CM mice and from inner white matter of control mice. AU, arbitrary units. (I) Differences in lactate in the spectra from hemorrhaged and nonhemorrhaged inner white matter from CM mice and from inner white matter of control mice. (J) Protein alterations in the spectra from granular layer tissue in CM mice and control mice. (K) Protein alterations in aggregated protein “hotspots” in the spectra from granular layer tissue of CM and control mice. Upward-facing arrows highlight spectral locations of decreased second-derivative band intensity, and downward facing arrows highlight spectral locations of increased second-derivative band intensity. (L) Brain and lung lactate levels in CM diseased and control mice (n = 5). Parasite DNA was not detected (ND) in the brain or lung of control mice. #Significant difference in brain lactate between CM and control mice. *Significant difference in lactate between the brain and lung of CM mice, or between the brain and lung of control mice. Significant difference in parasite DNA between the brain and lung of CM mice.

  • Fig. 3 XFM analysis of the elemental alterations in the cerebellum during murine CM.

    (A and B) XFM elemental maps (P, S, Cl, K, Ca, Fe, Cu, and Zn) collected with a 5-μm step size of the cerebellum of control (A) and CM (B) mice. H&E histology is presented for the entire cerebellum and at sites of healthy and hemorrhaged vasculature. IL, inner layer (white matter); GL, granular layer (gray matter). Arrows indicate the location of healthy vasculature in controls (black) and hemorrhaged (white) vasculature in ECM. (C and D) High–spatial resolution XFM elemental maps (P, S, Cl, K, Ca, Fe, Cu, and Zn) collected with a 0.5-μm step size of the cerebellum of control (C) and CM (D) mice. H&E histology is presented for the sites of healthy and hemorrhaged vasculature, indicated by black arrows. Note that high relative Fe content is only observed within the microvessel in the sham animal (similar to the preliminary investigation, fig. S8), whereas high Fe is observed throughout the white matter in the CM mouse, with low Fe observed in the white matter surrounding the vessel. IL, inner layer (white matter); GL, granular layer (gray matter).

  • Fig. 4 XFM elemental concentrations showing a significant difference in the P and Fe concentrations observed in hemorrhaged white matter tissue in CM mice, relative to healthy molecular layer, granular layer, and white matter tissue in control mice (n = 5).

    *Significant difference relative to control tissue. IL, inner layer (white matter); GL, granular layer (gray matter); ML, molecular layer; HM, hemorrhaged tissue.

  • Table 1 Differences in relative content of lipids, α helix protein, aggregated β sheet protein, and lactate as determined from second derivatives of FTIR band intensities for hemorrhaged inner white matter (WM HM), nonhemorrhaged inner white matter (WM), granular layer (GL), and molecular layer (ML) from CM and sham, mild malaria anemia (MMA), and severe malaria anemia (SMA) mice.

    Bold indicates a statistically significant difference (P < 0.05), relative to sham animals.

    Sample1742 cm−1 (lipid)1656 cm−1 (α helix protein)1625 cm−1 (aggregated β sheet protein)1127 cm−1 (lactate)
    CM WM HMAverage−2.5 × 10−4−2.6 × 10−32.3 × 10−41.3 × 10−5
    SD1.1 × 10−53.5 × 10−43.0 × 10−52.0 × 10−6
    CM WMAverage−6.6 × 10−4−1.8 × 10−33.0 × 10−46.6 × 10−5
    SD4.5 × 10−53.5 × 10−49.8 × 10−63.3 × 10−5
    Control (sham) WMAverage−7.2 × 10−4−1.8 × 10−33.6 × 10−49.1 × 10−5
    SD5.2 × 10−51.6 × 10−45.9 × 10−51.9 × 10−5
    Control (MMA) WMAverage−6.9 × 10−4−1.8 × 10−33.3 × 10−48.7 × 10−5
    SD2.5 × 10−52.6 × 10−42.7 × 10−51.4 × 10−5
    Control (SMA) WMAverage−7.0 × 10−4−1.8 × 10−33.4 × 10−48.6 × 10−5
    SD3.5 × 10−53.0 × 10−44.3 × 10−51.3 × 10−5
    CM GLAverage−2.4 × 10−4−2.0 × 10−32.5 × 10−46.0 × 10−5
    SD2.2 × 10−51.7 × 10−41.9 × 10−56.6 × 10−6
    Control (sham) GLAverage−2.4 × 10−4−2.1 × 10−33.0 × 10−45.0 × 10−5
    SD2.5 × 10−52.7 × 10−48.8 × 10−62.0 × 10−5
    Control (MMA) GLAverage−2.4 × 10−4−2.1 × 10−32.9 × 10−46.0 × 10−5
    SD3.0 × 10−52.7 × 10−41.5 × 10−53.3 × 10−5
    Control (SMA) GLAverage−2.6 × 10−4−2.1 × 10−32.7 × 10−45.5 × 10−5
    SD2.9 × 10−52.7 × 10−49.8 × 10−62.5 × 10−5
    CM MLAverage−1.6 × 10−4−2.1 × 10−32.7 × 10−44.0 × 10−5
    SD4.0 × 10−41.9 × 10−42.5 × 10−52.9 × 10−6
    Control (sham) MLAverage−3.5 × 10−5−2.1 × 10−32.9 × 10−44.1 × 10−5
    SD3.4 × 10−41.9 × 10−44.2 × 10−56.2 × 10−6
    Control (MMA) GLAverage−4.1 × 10−4−2.1 × 10−33.0 × 10−44.1 × 10−5
    SD7.5 × 10−52.0 × 10−44.2 × 10−53.0 × 10−6
    Control (SMA) MLAverage−2.3 × 10−4−2.2 × 10−32.7 × 10−43.8 × 10−5
    SD3.9 × 10−41.6 × 10−41.1 × 10−55.7 × 10−6

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/1/11/e1500911/DC1

    Text

    Fig. S1. Validation of second-derivative band intensity at 1127 cm−1 in the FTIR spectra as a marker for relative lactate concentration.

    Fig. S2. Validation of second-derivative band intensity at 1627 cm−1 in the FTIR spectra as a marker for relative concentration of aggregated β sheet proteins and, therefore, protein oxidation.

    Fig. S3. Correlation of FTIR maps of lipid distribution with H&E histology.

    Fig. S4. HCA analysis of FTIR maps distinguishes four distinct regions: the molecular layer, granular layer, inner white matter, and hemorrhaged white matter.

    Fig. S5. Principal components analysis of the average spectra for tissue layers determined from the HCA.

    Fig. S6. Synchrotron radiation–based (SR) FTIR mapping of cerebellum tissue to identify Mie scattering and electric field standing wave spectral features.

    Fig. S7. SR x-ray fluorescence elemental maps (2-μm step size) of healthy cerebellum blood vessels, showing location of Fe (white arrow) within the wall of the blood vessel.

    Fig. S8. PIXE elemental maps of healthy and hemorrhaged cerebellum blood vessels.

    Fig. S9. Resonance Raman spectra (514-nm excitation) collected from hemorrhaged tissue and dried red blood cells, showing characteristic enhanced intensity of hemoglobin bands.

    References (7394)

  • Supplementary Materials

    This PDF file includes:

    • Text
    • Fig. S1. Validation of second-derivative band intensity at 1127 cm−1 in the FTIR spectra as a marker for relative lactate concentration.
    • Fig. S2. Validation of second-derivative band intensity at 1627 cm−1 in the FTIR spectra as a marker for relative concentration of aggregated β sheet proteins and, therefore, protein oxidation.
    • Fig. S3. Correlation of FTIR maps of lipid distribution with H&E histology.
    • Fig. S4. HCA analysis of FTIR maps distinguishes four distinct regions: the molecular layer, granular layer, inner white matter, and hemorrhaged white matter.
    • Fig. S5. Principal components analysis of the average spectra for tissue layers determined from the HCA.
    • Fig. S6. Synchrotron radiation–based (SR) FTIR mapping of cerebellum tissue to identify Mie scattering and electric field standing wave spectra features.
      Fig. S7. SR x-ray fluorescence elemental maps (2-μm step size) of healthy cerebellum blood vessels, showing location of Fe (white arrow) within the wall of the blood vessel.
    • Fig. S8. PIXE elemental maps of healthy and hemorrhaged cerebellum blood vessels.
    • Fig. S9. Resonance Raman spectra (514-nm excitation) collected from hemorrhaged tissue and dried red blood cells, showing characteristic enhanced intensity of hemoglobin bands.
    • References (73–94)

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