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1.
Figure 4

Figure 4. Colocalization of Ngb, Orexin-A and c-FOS immunoreactivity after hypoxia.. From: Neuroglobin-Deficiency Exacerbates Hif1A and c-FOS Response, but Does Not Affect Neuronal Survival during Severe Hypoxia In Vivo.

Immunofluorescence staining of lateral hypothalamus from wt (A–B) and Ngb-null (C–D) mice after 90 minutes of hypoxia (Orexin-A (Hcrt) – green, c-FOS – red, Ngb – blue). High degree of co-expression of Orexin-A and Ngb-IR (white arrows in B) is observed in wt mice whereas c-FOS is hardly detectable. In Ngb-null mice, most of the Orexin-A-positive neurons are found to co-express c-FOS-IR (white arrows in D). Note the absence of Ngb-IR in the Ngb-null mouse. Scale bar 50 µm.

Christian Ansgar Hundahl, et al. PLoS One. 2011;6(12):e28160.
2.
Figure 2

Figure 2. The effect of hypoxia on the viability of neurons expressing surrogate markers of Ngb in wt and Ngb-deficient mice.. From: Neuroglobin-Deficiency Exacerbates Hif1A and c-FOS Response, but Does Not Affect Neuronal Survival during Severe Hypoxia In Vivo.

A) the number of Orexin-IR neurons in the lateral hypothalamus of naive wt and Ngb-null mice during normoxia and after 24 and 48 hours of hypoxia. No significant difference in the number of Orexin neurons was observed between the two genotypes (n = 3–6, p>0.05). B–C) the number of Cygb-IR neurons in PPTg (B) and LDTg (C) of wt (black bars) and Ngb-null (white bars) in naive mice and mice exposed to 24 h and 48 h of hypoxia. No significant difference in the number of Cygb-IR neurons was observed between the two genotypes (n = 3–6, p>0.05). Abbreviations: pedunculopontine tegmental nucleus (PPTg); laterodorsal tegmental nucleus (LDTg).

Christian Ansgar Hundahl, et al. PLoS One. 2011;6(12):e28160.
3.
Figure 5

Figure 5. Differential gene expression in response to hypoxia.. From: Neuroglobin-Deficiency Exacerbates Hif1A and c-FOS Response, but Does Not Affect Neuronal Survival during Severe Hypoxia In Vivo.

A–B: Differential expression (p<0.05) of several well-known hypoxia-responsive genes after acute (A) and 24 hours (B) hypoxia according to Affymetrix Mouse Gene 1.0 ST array. Bars represent differential expression between hypoxic and normoxic mice of the respective genotype based on the difference in the mean relative rank of the differentially expressed probes (wt mice - white bars, Ngb-null mice - gray bars). C–D: Genes with most reliable differential expression in response to acute (C) and 24-hour (D) hypoxia based on the product of differential expression p-values in both genotypes. Genes are ordered from left to right by the ascending product of differential expression p-values (i.e. decreasing reliability of differential expression). Bars represent differential expression between hypoxic and normoxic mice of the respective genotype based on the difference in the mean relative rank of the differentially expressed probes (wt mice - white bars, Ngb-null mice - gray bars).

Christian Ansgar Hundahl, et al. PLoS One. 2011;6(12):e28160.
4.
Figure 6

Figure 6. Functional annotation of differentially expressed genes in response to hypoxia.. From: Neuroglobin-Deficiency Exacerbates Hif1A and c-FOS Response, but Does Not Affect Neuronal Survival during Severe Hypoxia In Vivo.

Hypoxia-dependent regulation of gene expression in wt and Ngb-null mice in relation to known molecular pathways. Lists of genes that were differentially expressed between normoxic and acute hypoxic (90 minutes) or 24 hours hypoxic mice were subjected to functional annotation with g:Profiler [23]. Green background represents up-regulation and blue background represents down-regulation of the enriched pathway (p<0.001). The number of differentially expressed genes and the total number of genes in the pathway are indicated below the pathway's name. Due to space limitations, only extensively regulated (at least 30% of the pathway members are differentially expressed) and non-redundant (the pathways are represented by different sets of genes) pathways are presented.

Christian Ansgar Hundahl, et al. PLoS One. 2011;6(12):e28160.
5.
Figure 3

Figure 3. Induction of c-FOS immunoreactivity in response to hypoxia.. From: Neuroglobin-Deficiency Exacerbates Hif1A and c-FOS Response, but Does Not Affect Neuronal Survival during Severe Hypoxia In Vivo.

A–B) c-FOS-IR in the anterior forebrain of normoxic (A) and hypoxic (B) wt mice. The black square inserts of the cerebral cortex are shown in higher magnifications in A1 and B1. A modest induction of c-FOS-IR is apparent in hypoxic wt mice. C–D) c-FOS-IR in the anterior forebrain of naive (C) and hypoxic (D) Ngb-null mice. A substantial hypoxia-dependent increase of c-FOS-IR was observed in Ngb-null mice (D and D1) when compared to both naive Ngb-null (C and C1) and hypoxic wt (B and B1) mice. Similar genotype-dependent differences in c-FOS induction were observed in the posterior forebrain of wt (E, F) and Ngb-null mice (G, H). The black square inserts of the amygdaloid complex are magnified in E1H1. I–J) Ngb-IR in the anterior (I) and posterior forebrain (J) of naive wt mice. The black square inserts of the cerebral cortex and the amygdaloid complex are shown in higher magnifications in I1 and J1, respectively. Abbreviations: anterior commissure (ac); central amygdaloid nucleus, capsular part (CeC); caudate putamen (CPu); hippocampus (Hipp); medial preoptic area (MPA); optic tract (opt); piriform cortex (Pir). Scale bar 50 µm.

Christian Ansgar Hundahl, et al. PLoS One. 2011;6(12):e28160.
6.
Figure 1

Figure 1. Creation of the Ngb-deficient mouse strain.. From: Neuroglobin-Deficiency Exacerbates Hif1A and c-FOS Response, but Does Not Affect Neuronal Survival during Severe Hypoxia In Vivo.

A) the location of LoxP sites and Neo-cassette introduced in the Ngb gene by homologous recombination. B) immunohistochemical staining of hypothalamic tissue sections from wt and Ngb-null mice using a guinea pig anti-Ngb antibody. Strong Ngb immunoreactivity (IR) is observed in wt mice whereas IR was abolished in Ngb-null mice. C) Western blotting of Ngb-null (−/−), heterozygote (+/−) and wt (+/+) brains confirmed the absence of Ngb protein in Ngb-null mice. Recombinant Ngb (Rec Ngb) was used as a positive control. Actin was used as a loading control. D) Southern blot analysis for the validation of the heterozygous Neo-excised floxed mice. A 3′ external probe was used to confirm correct recombination and excision of Neo from the floxed allele. Predicted size of specific hybridization targets after digesting genomic DNA with BamHI-SpeI: wild-type allele 11153 bp, recombined allele 7765 bp, floxed (Flp-mediated Neo-excised) 9457 bp. Mice 16035 and 15716 are both positive for the Flp mediated Neo-deleted conditional knock-out Ngb allele. Note that mouse No. 16035 is a mosaic of Neo-excised and non-excised cells as indicated by the very weak signal from the nonexcised recombined allele. Mouse No. 15716 was used for the generation of the constitutive Ngb knockout strain. Abbreviations: 1 kb DNA-Ladder (NEB) (M); wild-type (wt); heterozygous Neo-excised floxed mice (16035 and 15716).

Christian Ansgar Hundahl, et al. PLoS One. 2011;6(12):e28160.

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