The deleterious effect of crossfostering in rat pups on hypoxic-ischemic injury tolerance and hypothermic neuroprotection

We study the effect of hypothermia (HT) following hypoxic-ischemic (HI) brain injury in postnatal day 7 (P7) rats. In 2015, new European Union animal transport regulations prompted a change in practice at the breeding facility, which henceforth crossfostered P3 litters to P8 older lactating dam prior to transportation. It is generally assumed that crossfostering does not significantly affect the experimental results. The aim of this study was to examine whether crossfostering affects our model consistency by modifying injury susceptibility and hypothermic neuroprotection. We analysed 219 pups (56 litters) from 11 experiments conducted between 2013 and 2015: 73 non-crossfostered and 146 crossfostered pups. At P7, all pups underwent unilateral common carotid artery ligation followed by 50min of hypoxia (8% O2, 36°C). Immediately after this mild insult, the pups were randomised to post-insult normothermia (NT) or HT treatment. Pups were culled at P14. Injury was assessed by area loss of the ipsilateral hemisphere and histopathology scoring of hippocampus, cortex, thalamus, and basal ganglia. Crossfostered pups had double the injury compared to non-crossfostered pups irrespective of treatment group. Hypothermic neuroprotection was statistically significant, but with a smaller and less consistent effect in crossfostered pups (relative neuroprotection 16% vs. 31% in non-crossfostered). These results demonstrate hypothermic neuroprotection following a mild HI insult. A representative subset of 41 animals were also assessed for evidence of microglial reactivity, however no detectable difference in microglial reactivity was observed between any of the groups. In conclusion, crossfostering alters outcomes in our established model through reduced insult tolerance and variable neuroprotection. Crossfostering as a common breeding practice is a largely unexplored variable in animal research that may result in invalid research conclusions if inadequately adjusted for by larger group sizes. As a result, crossfostering is likely to be inconsistent with the principles of replacement, reduction, and refinement.

Hypothermia Suppresses Uncoupling of Oxidative- Phosphorylation after Neonatal Cerebral Hypoxia-Ischemia

Hypothermia is the best available therapy for neonatal hypoxia ischemia (HI) brain injury, but its primary mechanisms remain uncertain. We hypothesize that HI induces, whereas hypothermia represses, uncoupling of oxidative phosphorylation (OXPHOS), an increase of the cerebral metabolic rate of oxygen (CMRO2) despite reduction of the mitochondrial energy output. We used a multiparametric photoacoustic microscopy (PAM) system to compare the effects of HI and post HI hypothermic treatment on CMRO2 in awake 10 day old (P10) mice. Here we show that hypoxia (10% O2) elevated CMRO2, but the addition of unilateral carotid artery ligation suppressed CMRO2 and sparked a rapid overshoot of post HI CMRO2 in the ipsilateral cerebral cortex for at least 2 hours. The post HI surge of CMRO2 was linked to an increase of mitochondrial oxygen consumption and superoxide outburst, despite reduction of the mitochondrial membrane potential. Notably, post HI hypothermia blocked the surge of superoxide and CMRO2, primarily by limiting oxygen extraction fraction (OEF), leading to better preservation of adenosine triphosphate (ATP), creatine (Cr) and N acetylaspartate (NAA) after HI. Mice that did not receive hypothermia exhibited ~80% reduction of CMRO2 at 24 h post HI, coupled to a large cortical infarction. These results suggest that mitigation of post HI uncoupling of OXPHOS is an early and/or pivotal effect of hypothermia. Further, optical measurement of CMRO2 may be a sensitive and noninvasive method to monitor brain damage in hypoxic ischemic encephalopathy (HIE).

Knockdown of IL-6 Plays a Crucial Role in Protection of Hypoxia-Ischemia in Neonatal Rats via Inhibiting Casp3 and BAX Signaling Pathway

Background: The effect of interleukin-6 (IL-6) knockdown on hypoxia-ischemia (HI) of neonatal rat models was investigated to explore the underlying molecular regulation mechanism. Methods: To establish the HI model, we treated 7days postnatal Sprague-Dawley (SD) rats with the right carotid artery ligation and had them exposed to the environment of 8% oxygen and 92% nitrogen for 2 h, respectively. Then, the neurologic function and morphology changes were assessed. Subsequently, IL-6 siRNA lentivirus was injected into cerebral cortex motor area 2 days before HI; meanwhile, the interference efficiency was detected by quantitative real-time polymerase chain reaction (QRT-PCR) and Western blot. Immunofluorescence staining of Glial fibrillary acidic protein (GFAP), Hexaribonucleotide Binding Protein-3 (NeuN) and IL-6 were used to identify the location and interference effect of IL-6. In order to further research the underlying mechanisms, the expressions of downstream molecular including Bcl-2-associated X protein (BAX) and Casp3 were examined following IL-6 up-regulation by QRT-PCR.Results: It was found that both the growth of cortical neurons and the length of axon were promoted after IL-6 interference, and the cell apoptosis was decreased. In addition, the expression of BAX and Casp3 were closely associated with IL-6. Conclusions: The present findings confirmed that the decreased IL-6 improves the deficiencies in neurologic function and morphology induced by HI, and the potential mechanism may be closely related with the regulation of Casp3 and BAX.

Partial endothelial-to-mesenchymal transition (EndMT) contributes to lumen re-organization after carotid artery ligation

Endothelial-to-mesenchymal transition (EndMT) is a fundamental process in vascular remodeling. Carotid artery ligation is commonly used for induction of neointima formation and vessel stenosis; however, the precise regulatory mechanism of vascular remodeling is not entirely understood. In this study, we showed that resident endothelial cells (ECs) are the origin of neointima cells and ECs transiently expressed CD45 in the early stage of neointima formation accompanied by increased expression of EndMT markers. In vitro, CD45-positive EndMT was induced by stabilization of HIF-1α with cobalt chloride or VHL inhibitor in human primary ECs, which mimicked the hypoxic condition of ligated artery, and promoted the formation of integrin α11-SHARPIN complex. Notably, a CD45 phosphatase inhibitor disrupted this complex, thereby destabilizing cell-cell junctions. These results suggest that the CD45 activity is required for the retention of an EC phenotype and cell-cell junctions during EndMT (termed partial EndMT). We thus propose a novel mechanism of partial EndMT that contributes to lumen re-organization during vascular injury.

Inhibitory Effect of a Glutamine Antagonist on Proliferation and Migration of VSMCs via Simultaneous Attenuation of Glycolysis and Oxidative Phosphorylation

Excessive proliferation and migration of vascular smooth muscle cells (VSMCs) contribute to the development of atherosclerosis and restenosis. Glycolysis and glutaminolysis are increased in rapidly proliferating VSMCs to support their increased energy requirements and biomass production. Thus, it is essential to develop new pharmacological tools that regulate metabolic reprogramming in VSMCs for treatment of atherosclerosis. The effects of 6-diazo-5-oxo-L-norleucine (DON), a glutamine antagonist, have been broadly investigated in highly proliferative cells; however, it is unclear whether DON inhibits proliferation of VSMCs and neointima formation. Here, we investigated the effects of DON on neointima formation in vivo as well as proliferation and migration of VSMCs in vitro. DON simultaneously inhibited FBS- or PDGF-stimulated glycolysis and glutaminolysis as well as mammalian target of rapamycin complex I activity in growth factor-stimulated VSMCs, and thereby suppressed their proliferation and migration. Furthermore, a DON-derived prodrug, named JHU-083, significantly attenuated carotid artery ligation-induced neointima formation in mice. Our results suggest that treatment with a glutamine antagonist is a promising approach to prevent progression of atherosclerosis and restenosis.

Contribution of PDGFRα-positive cells in maintenance and injury responses in mouse large vessels

The maladaptive remodeling of vessel walls with neointima formation is a common feature of proliferative vascular diseases. It has been proposed that neointima formation is caused by the dedifferentiation of mature smooth muscle cells (SMCs). Recent evidence suggests that adventitial cells also participate in neointima formation; however, their cellular dynamics are not fully understood. In this study, we utilized a lineage tracing model of platelet-derived growth factor receptor alpha (PDGFRa) cells and examined cellular behavior during homeostasis and injury response. PDGFRa marked adventitial cells that were largely positive for Sca1 and a portion of medial SMCs, and both cell types were maintained for 2 years. Upon carotid artery ligation, PDGFRa-positive (+) cells were slowly recruited to the neointima and exhibited an immature SMC phenotype. In contrast, in a more severe wire denudation injury, PDGFRa+ cells were recruited to the neointima within 14 days and fully differentiated into SMCs. Under pressure overload induced by transverse aortic constriction, PDGFRa+ cells developed marked adventitial fibrosis. Taken together, our observations suggest that PDGFRa+ cells serve as a reservoir of adventitial cells and a subset of medial SMCs and underscore their context-dependent response to vascular injuries.

Artemin Is Upregulated by TrkB Agonist and Protects the Immature Retina Against Hypoxic-Ischemic Injury by Suppressing Neuroinflammation and Astrogliosis

Hypoxic-ischemia (HI) is a major cause of acquired visual impairment in children from developed countries. Previous studies have shown that systemic administration of 7,8-dihydroxyavone (DHF), a selective tropomyosin receptor kinase B (TrkB) agonist, provides long-term neuroprotection against HI injury in an immature retina. However, the target genes and the mechanisms of the neuroprotective effects of TrkB signaling are not known. In the present study, we induced an HI retinal injury through unilateral common carotid artery ligation followed by 8% oxygen for 2 h in P7 rat pups. DHF was administered intraperitoneally 2 h before and 18 h after the HI injury. A polymerase chain reaction (PCR) array was used to identify the target genes upregulated after the DHF treatment, which was then confirmed with quantitative real-time reverse transcriptase PCR and a western blot. Effects of the downstream mediator of DHF were assessed using an intravitreal injection of neutralizing antibody 4 h after DHF administration (24 h after HI). Meanwhile, the target protein was injected into the vitreous 24 h after HI to validate its protective effect when exogenously supplemented. We found that systemic DHF treatment after HI significantly increased the expression of the artemin (ARTN) gene and protein at P8 and P10, respectively. The neuroprotective effects of DHF were inhibited after the ARTN protein blockade, with an increase in neuroinflammation and astrogliosis. ARTN treatment showed long-term protection against HI injury at both the histopathological and functional levels. The neuroprotective effects of ARTN were related to a decrease in microglial activation at P17 and attenuation of astrogliosis at P29. ARTN enhances phosphorylation of RET, ERK, and JNK, but not AKT or p38 in the immature retina. Altogether, these results suggest that the neuroprotective effect of a TrkB agonist is partially exerted through a mechanism that involves ARTN because the protective effect is ameliorated by ARTN sequestration. ARTN treatment after HI injury protects the immature retina by attenuating late neuroinflammation and astrogliosis in the immature retina relating to the ARTN/RET/JNK/ERK signaling pathway. ARTN may be a strategy by which to provide long-term protection in the immature retina against HI injury.