Functional Real-Time Optoacoustic Imaging of Middle Cerebral Artery Occlusion in Mice

Background and Purpose

Longitudinal functional imaging studies of stroke are key in identifying the disease progression and possible therapeutic interventions. Here we investigate the applicability of real-time functional optoacoustic imaging for monitoring of stroke progression in the whole brain of living animals.

Materials and Methods

The middle cerebral artery occlusion (MCAO) was used to model stroke in mice, which were imaged preoperatively and the occlusion was kept in place for 60 minutes, after which optoacoustic scans were taken at several time points.

Results

Post ischemia an asymmetry of deoxygenated hemoglobin in the brain was observed as a region of hypoxia in the hemisphere affected by the ischemic event. Furthermore, we were able to visualize the penumbra in-vivo as a localized hemodynamically-compromised area adjacent to the region of stroke-induced perfusion deficit.

Conclusion

The intrinsic sensitivity of the new imaging approach to functional blood parameters, in combination with real time operation and high spatial resolution in deep living tissues, may see it become a valuable and unique tool in the development and monitoring of treatments aimed at suspending the spread of an infarct area.     

Systemic levels contribute significantly to increased intraocular IGF-I, IGF-II and IGF-BP3 [correction of IFG-BP3] in proliferative diabetic retinopathy.

Increased intraocular levels of angiogenic growth factors such as insulin-like growth factor I (IGF-I) have been demonstrated in proliferative diabetic retinopathy (PDR). It is unclear whether increased leakage of the blood retina barrier or local synthesis primarily determine intraocular levels of IGFs in man, which is of special interest regarding possible therapeutic options with somatostatin analogues in PDR. This is the first study investigating parallelly serum and vitreous levels of IGF-I/II, IGF-BP3 and the liver-derived permeability marker albumin to determine in vivo the amount of circulation-derived intraocular IGFs. A control group without retinal proliferation and patients with PDR were compared. Levels of IGF-I/II, IGF-BP3 and albumin were determined by immunological methods. Vitreous levels of albumin were 2.2-fold elevated in patients with PDR (254.1 +/- 37.2mg/dl; n = 27; p = 0.0027) compared to controls (115.7 +/- 36.2mg/dl; n =10), whereas serum levels were slightly decreased in diabetes patients (5049 +/- 196 mg/dl vs. 4330 +/- 186 mg/dl; p = 0.0283). This was comparable to an increase of IGF-I/11 and IGF-BP3 in vitreous from PDR patients (IGF-I: 2.3 +/- 1.1 ng/ml p = 0.005. IGF-II: 37.9 +/- 4.9 ng/ml; p = 0.0003. IGF-BP3: 97.9 +/- 26.9 ng/ml; p = 0.0001; n = 34) compared to controls (IGF-I: 0.7 +/- 0.1 ng/ml. IGF-II: 21.3 +/- 4.2 ng/ml. IGF-BP3: 31.3 +/- 4.9 ng/ml: n = 19). Serum levels did not differ significantly among the groups regarding IGF-I, II and IGF-BP3. Intraocular albumin and IGF-I levels calculated as percentage of the respective serum levels correlated significantly (r = 0.42; p = 0.012). This study demonstrates that influx of IGF-I, II and IGF-BP3 in PDR quantitatively parallels influx of the liver derived serum protein albumin suggesting that leakage of the blood retina barrier and serum levels of IGF primarily determine intravitreal IGF levels rather than local synthesis. Suppression of systemic IGF levels by new, highly effective somatostatin-analogues therefore provides a promising approach to prevent PDR.     

Defective attachment of dermatosparactic fibroblasts to collagens I and IV

Attachment of fibroblasts from dermatosparactic sheep and cattle to collagenous substrates (types I and IV) is defective (30-50%) when compared with fibroblasts from normal or heterozygous animals. The difference was independent of the amount of substrate, incubation time and protein synthesis. No differences were observed in the binding to fibronectin or laminin. Reduced attachment to collagen can be partially restored by adding fibronectin. The polygonal morphology of dermatosparactic cells was, however, not altered by attachment and growth on dishes coated with different collagens or fibronectin. Reduced interaction with collagens could be due to changes in specific receptors and may represent a further pathological change in dermatosparactic animals.     

Delineation of a CpG phosphorothioate oligodeoxynucleotide for activating primate immune responses in vitro and in vivo

Oligodeoxynucleotides (ODN) containing unmethylated CpG dinucleotides within specific sequence contexts (CpG motifs) are detected, like bacterial or viral DNA, as a danger signal by the vertebrate immune system. CpG ODN synthesized with a nuclease-resistant phosphorothioate backbone have been shown to be potent Th1-directed adjuvants in mice, but these motifs have been relatively inactive on primate leukocytes in vitro. Moreover, in vitro assays that predict in vivo adjuvant activity for primates have not been reported. In the present study we tested a panel of CpG ODN for their in vitro and in vivo immune effects in mice and identified in vitro activation of B and NK cells as excellent predictors of in vivo adjuvant activity. Therefore, we tested >250 phosphorothioate ODN for their capacity to stimulate proliferation and CD86 expression of human B cells and to induce lytic activity and CD69 expression of human NK cells. These studies revealed that the sequence, number, and spacing of individual CpG motifs contribute to the immunostimulatory activity of a CpG phosphorothioate ODN. An ODN with a TpC dinucleotide at the 5' end followed by three 6 mer CpG motifs (5'-GTCGTT-3') separated by TpT dinucleotides consistently showed the highest activity for human, chimpanzee, and rhesus monkey leukocytes. Chimpanzees or monkeys vaccinated once against hepatitis B with this CpG ODN adjuvant developed 15 times higher anti-hepatitis B Ab titers than those receiving vaccine alone. In conclusion, we report an optimal human CpG motif for phosphorothioate ODN that is a candidate human vaccine adjuvant.     

Modulation of the Acetylcholine System in the Superior Cervical Ganglion of Rat: Effects of GABA and Hypoglossal Nerve Implantation After In Vivo GABA Treatment

gamma-Aminobutyric acid (GABA) was applied to the superior cervical ganglion (SCG) of CFY rats in vitro and in vivo, with or without implantation of a hypoglossal nerve, to evaluate the effects of these experimental interventions on the acetylcholine (ACh) system, which mainly serves the synaptic transmission of the preganglionic input. Long-lasting GABA microinfusion into the SCG in vivo apparently resulted in a "functional denervation." This treatment reduced the acetylcholinesterase (AChE; EC 3.1.1.7) activity by 30% (p less than 0.01) and transiently increased the number of nicotinic acetylcholine receptors, but had no significant effect on the choline acetyltransferase (acetyl-coenzyme A:choline-O-acetyltransferase; EC 2.3.1.6) activity, the ACh level, or the number of muscarinic acetylcholine receptors. The relative amounts of the different molecular forms of AChE did not change under these conditions. In vivo GABA application to the SCG with a hypoglossal nerve implanted in the presence of intact preganglionic afferent synapses exerted a significant modulatory effect on the AChE activity and its molecular forms. The "hyperinnervation" of the ganglia led to increases in the AChE activity (to 142.5%, p less than 0.01) and the 16S molecular form (to 200%, p less than 0.01). It is concluded that in vivo GABA microinfusion and GABA treatment in the presence of additional cholinergic synapses has a modulatory effect on the elements of the ACh system in the SCG of CFY rats.     

AlgR-binding sites within the algD promoter make up a set of inverted repeats separated by a large intervening segment of DNA.

Activation of algD by AlgR is essential for mucoidy, a virulence factor expressed by Pseudomonas aeruginosa in cystic fibrosis. Two AlgR-binding sites, RB1 and RB2, located far upstream from the algD mRNA start site, are essential for the high-level activity of algD. However, the removal of RB1 and RB2 does not completely abolish inducibility of algD in response to environmental signals. In this work, a third binding site for AlgR, termed RB3, near the algD mRNA start site was characterized. Deletion of RB3 abrogated both the AlgR-binding ability and the residual inducibility of the algD promoter. DNase I footprinting analysis of RB3 resulted in a protection pattern spanning nucleotides -50 to -30. Eight of 10 residues encompassing a continuous region of protection within RB3 (positions -45 to -36) matched in the inverted orientation the conserved core sequence (ACCGTTCGTC) of RB1 and RB2. Quantitative binding measurements of AlgR association with RB1, RB2, and RB3 indicated that AlgR had significantly lower affinity for RB3 than for RB1 and RB2, with differences in the free energy of binding of 1.05 and 0.93 kcal/mol (4.39 and 3.89 kJ/mmol), respectively. Altering the core of RB2 to match the core of RB3 significantly reduced AlgR binding. Conversely, changing the core of RB3 to perfectly match the core of RB2 (mutant site termed RB3*) improved AlgR binding, approximating the affinity of RB2. RB3*, in the absence of the far upstream sites, showed an increase in activity, approaching the levels observed with the full-size algD promoter. Changing 4 nucleotides in two different combinations within the core of RB3 abolished the binding of AlgR to this site and resulted in a significant reduction of promoter activity in the presence of the far upstream sites. Thus, (i) the core sequence is essential for AlgR binding; (ii) the three binding sites, RB1, RB2, and RB3, are organized as an uneven palindrome with symmetrical sequences separated by 341 and 417 bp; and (iii) all three sites participate in algD activation.     

Enzymatic peptide synthesis by the recombinant proline-specific endopeptidase from Flavobacterium meningosepticum and its mutationally altered Cys-556 variant

Proline-specific endopeptidase (PSE) (EC 3.4.21.26) was investigated for its potential as a catalyst in peptide synthesis. Using an activated peptide ester or a peptide amide as the acyl component, the enzyme catalyzed kinetically controlled aminolysis and transpeptidation respectively, with various amino acid amides as acyl acceptors. To a certain extent the nucleophile preference reflected the amino acid preference in the S₁-position of the enzyme in peptide hydrolysis: the highest fractions of aminolysis were obtained using amino acid amides with hydrophobic side-chains (e.g. Leu-NH₂, Phe-NH₂). PSE also catalyzed the thermodynamically controlled condensation of short peptides with a free carboxyterminus and various amino acid amides. This enabled us to examine the acceptance of different acyl components in the substrate-binding site of the enzyme with regard to their amino acid composition: In the S₁ position proline was clearly favored, but alanine was also accepted, whereas the S₂ subsite accepted various amino acids rather unspecifically. Since PSE was shown to be extremely sensitive against water-miscible organic solvents, an alternative approach was used to increase yields in enzymatic peptide synthesis: a derivative of PSE in which the catalytic Ser-556 is converted to a Cys was constructed by protein engineering. This mutant (PSE_cys) exhibited a dramatically increased peptide ligase activity in aqueous solution.