Very Delayed Remote Ischemic Post-conditioning Induces Sustained Neurological Recovery by Mechanisms Involving Enhanced Angioneurogenesis and Peripheral Immunosuppression Reversal

Ischemic conditioning is defined as a transient and subcritical period of ischemia integrated in an experimental paradigm that involves a stimulus of injurious ischemia, activating endogenous tissue repair mechanisms that lead to cellular protection under pathological conditions like stroke. Whereas ischemic pre-conditioning is irrelevant for stroke treatment, ischemic post-conditioning, and especially non-invasive remote ischemic post-conditioning (rPostC) is an innovative and potential strategy for stroke treatment. Although rPostC has been shown to induce neuroprotection in stroke models before, resulting in some clinical trials on the way, fundamental questions with regard to its therapeutic time frame and its underlying mechanisms remain elusive. Hence, we herein used a model of non-invasive rPostC of hind limbs after cerebral ischemia in male C57BL6 mice, studying the optimal timing for the application of rPostC and its underlying mechanisms for up to 3 months. Mice undergoing rPostC underwent three different paradigms, starting with the first cycle of rPostC 12 h, 24 h, or 5 days after stroke induction, which is a very delayed time point of rPostC that has not been studied elsewhere. rPostC as applied within 24 h post-stroke induces reduction of infarct volume on day three. On the contrary, very delayed rPostC does not yield reduction of infarct volume on day seven when first applied on day five, albeit long-term brain injury is significantly reduced. Likewise, very delayed rPostC yields sustained neurological recovery, whereas early rPostC (i.e., <24 h) results in transient neuroprotection only. The latter is mediated via heat shock protein 70 that is a well-known signaling protein involved in the pathophysiological cellular cascade of cerebral ischemia, leading to decreased proteasomal activity and decreased post-stroke inflammation. Very delayed rPostC on day five, however, induces a pleiotropic effect, among which a stimulation of angioneurogenesis, a modulation of the ischemic extracellular milieu, and a reversal of the stroke-induced immunosuppression occur. As such, very delayed rPostC appears to be an attractive tool for future adjuvant stroke treatment that deserves further preclinical attention before large clinical trials are in order, which so far have predominantly focused on early rPostC only

Multiscale x-ray phase-contrast tomography in a mouse model of transient focal cerebral ischemia

Cerebral ischemia is associated with a lack of oxygen and high-energy phosphates within the brain tissue, leading to irreversible cell injury. Visualizing these cellular injuries has long been a focus of experimental stroke research with application of immunohistochemistry as one of the standard approaches. It is, however, a destructive imaging technique with non-isotropic resolution, as only the two-dimensional tissue structure of a thin brain section is visualized using optical microscopy and specific stainings. Herein, we extend the structural analysis of mouse brain tissue after cerebral ischemia to the third dimension via microfocus computed tomography (µ-CT). Contrast of the weakly absorbing unstained brain tissue is enhanced by phase contrast. We show that recordings at two different magnifications and fields of view can be combined as a single approach for visualization of the associated structural alterations at isotropic resolution, from the level of the whole organ down to single cells

Quantification of Bile Acids in Cerebrospinal Fluid: Results of an Observational Trial

(1) Background: Bile acids, known as aids in intestinal fat digestion and as messenger molecules in serum, can be detected in cerebrospinal fluid (CSF), although the blood–brain barrier is generally an insurmountable obstacle for bile acids. The exact mechanisms of the occurrence, as well as possible functions of bile acids in the central nervous system, are not precisely understood. (2) Methods: We conducted a single-center observational trial. The concentrations of 15 individual bile acids were determined using an in-house LC-MS/MS method in 54 patients with various acute and severe disorders of the central nervous system. We analyzed CSF from ventricular drainage taken within 24 h after placement, and blood samples were drawn at the same time for the presence and quantifiability of 15 individual bile acids. (3) Results: At a median time of 19.75 h after a cerebral insult, the concentration of bile acids in the CSF was minute and almost negligible. The CSF concentrations of total bile acids (TBAs) were significantly lower compared to the serum concentrations (serum 0.37 µmol/L [0.24, 0.89] vs. 0.14 µmol/L [0.05, 0.43]; p = 0.033). The ratio of serum-to-CSF bile acid levels calculated from the respective total concentrations were 3.10 [0.94, 14.64] for total bile acids, 3.05 for taurocholic acid, 14.30 [1.11, 27.13] for glycocholic acid, 0.0 for chenodeoxycholic acid, 2.19 for taurochenodeoxycholic acid, 1.91 [0.68, 8.64] for glycochenodeoxycholic acid and 0.77 [0.0, 13.79] for deoxycholic acid; other bile acids were not detected in the CSF. The ratio of CSF-to-serum S100 concentration was 0.01 [0.0, 0.02]. Serum total and conjugated (but not unconjugated) bilirubin levels and serum TBA levels were significantly correlated (total bilirubin p = 0.031 [0.023, 0.579]; conjugated bilirubin p = 0.001 [0.193, 0.683]; unconjugated p = 0.387 [−0.181, 0.426]). No correlations were found between bile acid concentrations and age, delirium, intraventricular blood volume, or outcome measured on a modified Rankin scale. (4) Conclusions: The determination of individual bile acids is feasible using the current LC-MS/MS method. The results suggest an intact blood–brain barrier in the patients studied. However, bile acids were detected in the CSF, which could have been achieved by active transport across the blood–brain barrier

CCL11 differentially affects post-stroke brain injury and neuroregeneration in mice depending on age

CCL11 has recently been shown to differentially affect cell survival under various pathological conditions including stroke. Indeed, CCL11 promotes neuroregeneration in neonatal stroke mice. The impact of CCL11 on the adult ischemic brain, however, remains elusive. We therefore studied the effect of ectopic CCL11 on both adolescent (six-week) and adult (six-month) C57BL6 mice exposed to stroke. Intraperitoneal application of CCL11 significantly aggravated acute brain injury in adult mice but not in adolescent mice. Likewise, post-stroke neurological recovery after four weeks was significantly impaired in adult mice whilst CCL11 was present. On the contrary, CCL11 stimulated gliogenesis and neurogenesis in adolescent mice. Flow cytometry analysis of blood and brain samples revealed a modification of inflammation by CCL11 at subacute stages of the disease. In adolescent mice, CCL11 enhances microglial cell, B and T lymphocyte migration towards the brain, whereas only the number of B lymphocytes is increased in the adult brain. Finally, the CCL11 inhibitor SB297006 significantly reversed the aforementioned effects. Our study, for the first time, demonstrates CCL11 to be a key player in mediating secondary cell injury under stroke conditions. Interfering with this pathway, as shown for SB297006, might thus be an interesting approach for future stroke treatment paradigms

Evaluation of the therapeutic potential of anti-TLR4 mAb <i>in vivo</i> applied <i>i</i>.<i>a</i>..

<p>MTS510 mAb (<b>TLR4-Ab</b>), isotype control (<b>Iso</b>), or vehicle (<b>PBS</b>) was applied <i>i</i>.<i>a</i>. at the start of MCAO (45min) in a dose of 1μg/animal in each application. Besides application of isotype control antibody, a second control was performed by omission of any antibody and injection of vehicle only (<b>PBS</b>). The difference between direct (<b>A</b>) and indirect (<b>B</b>) infarct volumes at 48h of reperfusion represents brain swelling/edema (<b>C</b>). Neurological deficit (<b>D</b>) at 48h of reperfusion after 45min of MCAO was ranked according to a modified Bederson-score from 0 (no deficit) up to 4 (dead). Data are presented as single values (<i>scatter blots</i>) combined with the mean ± SD; neuroscore graph shows single values and the median (<b>***</b>p < 0.001; <b>**</b>p < 0.01; <b>*</b>p < 0.05; Mann-Whitney U test; number of animals in treatment groups: n_<i>iso</i> = 8, n_<i>PBS</i> = 10, n_{<i>TLR4Ab</i>} = 10).</p

Evaluation of the therapeutic potential of of anti-TLR4 mAb <i>in vivo</i> applied <i>i</i>.<i>p</i>..

<p>MTS510 mAb (<b>TLR4-Ab</b>), or isotype control antibody (<b>Iso</b>) were applied <i>i</i>.<i>p</i>. at the end of 45min MCAO and once again after 24h of reperfusion in a dose of 1μg/animal in each application. A second control was performed by omission of any antibody and thus pure injection of vehicle (<b>PBS</b>). A correction for edema (brain swelling) was applied by calculating the indirect infarct volume as the volume of the contralateral hemisphere minus the non-infarcted volume of the ipsilateral hemisphere. The difference between direct (<b>A</b>) and indirect (<b>B</b>) infarct volumes represents brain swelling (<b>C</b>). Neurological deficit (<b>D</b>) was ranked according to a modified Bederson-score from 0 (no deficit) to 4 (dead). Data are presented as single values (<i>scatter blots</i>) combined with the mean ± SD; neuroscore graph shows single values and median (n_<i>Iso</i> = 10; n_<i>PBS</i> = 10; n_{<i>TLR4Ab</i>} = 8).</p

Evaluation of inflammatory cell counts in whole hemispheres of <i>i</i>.<i>v</i>. vehicle, and anti-TLR4mAb treated wild-type mice at 48h after 45min MCAO.

<p>Total cell count in each hemisphere was determined by the use of flow cytometry. CD11b-positive cells (activated macrophages/microglia) were shown with their absolute cell counts (<b>A</b>) in each, ispilateral/ischemic (<b>ipsi</b>) and contralateral/non-ischemic (<b>contra</b>) hemisphere of PBS- or anti-TLR4mAb-treated mice; as well as with the ratio of induction in ischemic hemisphere when compared to contralateral/non-ischemic hemisphere (<b>B</b>). Fig 5C shows the number of neutrophils identified in whole ischemic and non-ischemic hemispheres of PBS and anti-TLR4-mAb treated mice (mean values ± SD; <b>*</b>p< 0.05; Mann-Whitney U; n_<i>PBS</i> = 6; n_{<i>TLR4Ab</i>} = 5).</p

Anti-mouse TLR4/MD2 antibody clone MTS510 suppresses TLR4-mediated immune response <i>in vitro</i>.

<p>Murine RAW264.7 macrophage cells were incubated with (or without) anti-TLR4/MD2 (clone MTS510) antibody for 30min before incubation of the cells for 2 days with 10ng/ml lipopolysaccharide (LPS). TNFα concentration in the supernatant was determined by the use of a bioassay with the L929 reporter cell line and supernatant (1:200 dilution). TNFα concentration is indicated in pg/ml and was determined with 4 measurements of three separate biological samples in each group (<b>control</b>: incubation without LPS and MTS510 antibody; <b>LPS</b>: incubation with LPS only; <b>LPS+TLR4</b>: incubation with MTS510 antibody before stimulation with LPS), data are present with the mean ± SD (<b>***</b>p< 0.0001 as determined by Mann-Whitney U test).</p

Evaluation of cells of the adaptive immune response at 48h after 45min MCAO in mice <i>i</i>.<i>v</i>. treated with anti-TLR4mAb or vehicle control.

<p>Through CD3- or CD45.B220-specific selection by the use of flow cytometry, the total T-cell count (<b>A</b>), and the total B-cell count (<b>B</b>) in whole ischemic (<b>ipsi</b>), and non-ischemic (<b>contra</b>) hemispheres of <i>i</i>.<i>v</i>. TLR4mAb treated (<b>TLR4-Ab</b>) and vehicle (<b>PBS</b>) treated mice was determined at 48h after 45min of MCAO (mean values ± SD; <b><i>*</i></b><i>p</i>< 0.05; Mann-Whitney U; n_<i>PBS</i> = 6; n_{<i>TLR4Ab</i>} = 5).</p