Expression of the cobalamin transporters cubam and MRP1 in the canine ileum-Upregulation in chronic inflammatory enteropathy.

Chronic inflammatory enteropathy (CIE) in dogs, a spontaneous model of human inflammatory bowel disease (IBD), is associated with a high rate of cobalamin deficiency. The etiology of hypocobalaminemia in human IBD and canine CIE remains unknown, and compromised intestinal uptake of cobalamin resulting from ileal cobalamin receptor deficiency has been proposed as a possible cause. Here, we evaluated the intestinal expression of the cobalamin receptor subunits, amnionless (AMN) and cubilin (CUBN), and the basolateral efflux transporter multi-drug resistance protein 1 (MRP1) in 22 dogs with CIE in comparison to healthy dogs. Epithelial CUBN and AMN levels were quantified by confocal laser scanning microscopy using immunohistochemistry in endoscopic ileal biopsies from dogs with (i) CIE and normocobalaminemia, (ii) CIE and suboptimal serum cobalamin status, (iii) CIE and severe hypocobalaminemia, and (iv) healthy controls. CUBN and MRP1 expression was quantified by RT-qPCR. Receptor expression was evaluated for correlation with clinical patient data. Ileal mucosal protein levels of AMN and CUBN as well as mRNA levels of CUBN and MRP1 were significantly increased in dogs with CIE compared to healthy controls. Ileal cobalamin receptor expression was positively correlated with age, clinical disease activity index (CCECAI) score, and lacteal dilation in the ileum, inversely correlated with serum folate concentrations, but was not associated with serum cobalamin concentrations. Cobalamin receptor downregulation does not appear to be the primary cause of hypocobalaminemia in canine CIE. In dogs of older age with severe clinical signs and/or microscopic intestinal lesions, intestinal cobalamin receptor upregulation is proposed as a mechanism to compensate for CIE-associated hypocobalaminemia. These results support oral supplementation strategies in hypocobalaminemic CIE patients.

Impact of 5-lipoxygenase inhibitors on the spatiotemporal distribution of inflammatory cells and neuronal COX-2 expression following experimental traumatic brain injury in rats.

The inflammatory response following traumatic brain injury (TBI) contributes to neuronal death with poor outcome. Although anti-inflammatory strategies were beneficial in the experimental TBI, clinical translations mostly failed, probably caused by the complexity of involved cells and mediators. We recently showed in a rat model of controlled cortical impact (CCI) that leukotriene inhibitors (LIs) attenuate contusion growth and improve neuronal survival. This study focuses on spatiotemporal characteristics of macrophages and granulocytes, typically involved in inflammatory processes, and neuronal COX-2 expression. Effects of treatment with LIs (Boscari/MK-886), started prior trauma, were evaluated by quantifying CD68(+), CD43(+) and COX-2(+) cells 24h and 72 h post-CCI in the parietal cortex (PC), CA3 region, dentate gyrus (DG) and visual/auditory cortex (v/aC). Correlations were applied to identify intercellular relationships. At 24h, untreated animals showed granulocyte invasion in all regions, decreasing towards 72 h. Macrophages increased from 24h to 72 h post-CCI in PC and v/aC. COX-2(+) neurones showed no temporal changes, except of an increase in the CA3 region at 72 h. Treatment reduced granulocytes at 24h in the pericontusional zone and hippocampus, and macrophages at 72 h in the PC and v/aC. COX-2 expression remained unaffected by LIs, except of time-specific changes in the DG (increase/decrease at 24/72 h). Interrelations confirmed concomitant cellular reactions beyond the initial trauma site. In conclusion, LIs attenuated the cellular inflammatory response following CCI. Future studies have to clarify region-specific effects and explore the potential of a clinically more relevant therapeutic approach applying LIs after CCI.

Long-lasting neuronal loss following experimental focal cerebral ischemia is not affected by combined administration of tissue plasminogen activator and hyperbaric oxygen.

Acute focal cerebral ischemia and consecutive energy failure are accompanied by neuronal death in regions with impaired cerebral blood flow. Several translational attempts of potential neuroprotective agents have failed, hence extended perspectives are required regarding the regional differences of neuronal impairment and glial involvement by using clinically relevant stroke models. This study aimed on neuronal loss following experimental focal cerebral ischemia, considering tissue plasminogen activator (tPA) as established treatment in stroke and hyperbaric oxygenation (HBO) as potential neuroprotective co-treatment. Wistar rats were subjected to embolic middle cerebral artery occlusion and underwent either treatment with tPA only, combined tPA+HBO, or no treatment. Neuronal impairment was assessed by Neuronal Nuclei (NeuN) staining in 4 ischemia-related areas and at 4 different time points after stroke induction (24hours, 7, 14 and 28 days). Additionally, spatial relationships between neuronal loss and gliosis were revealed by triple fluorescence staining of neurons, astrocytes and microglia, comparing the ipsi- and contra-lesional hemisphere. Analyzing the ischemic injury in general, a shell-like distribution of neuronal damage was observed, starting in the ischemic core and diminishing over the general ischemic area to the ischemic border zone and the primary non-affected area. This pattern remained detectable up to 4weeks after ischemia induction. Surprisingly, tPA and tPA+HBO did not markedly affect the post-ischemic course of neuronal impairment. Further studies are needed to investigate the effects of treatment with tPA or potential neuroprotective agents on neuronal integrity, with emphasis on the separation of intact neurons from those undergoing apoptosis or necrosis.

Early outcome and blood-brain barrier integrity after co-administered thrombolysis and hyperbaric oxygenation in experimental stroke.

BACKGROUND: After promising results in experimental stroke, normobaric (NBO) or hyperbaric oxygenation (HBO) have recently been discussed as co-medication with tissue plasminogen activator (tPA) for improving outcome. This study assessed the interactions of hyperoxia and tPA, focusing on survival, early functional outcome and blood-brain barrier (BBB) integrity following experimental stroke. METHODS: Rats (n = 109) underwent embolic middle cerebral artery occlusion or sham surgery. Animals were assigned to: Control, NBO (60-minute pure oxygen), HBO (60-minute pure oxygen at 2.4 absolute atmospheres), tPA, or HBO+tPA. Functional impairment was assessed at 4 and 24 hours using Menzies score, followed by intravenous application of FITC-albumin as a BBB permeability marker, which was allowed to circulate for 1 hour. Further, blood sampling was performed at 5 and 25 hours for MMP-2, MMP-9, TIMP-1 and TIMP-2 concentration. RESULTS: Mortality rates did not differ significantly between groups, whereas functional improvement was found for NBO, tPA and HBO+tPA. NBO and HBO tended to stabilize BBB and to reduce MMP-2. tPA tended to increase BBB permeability with corresponding MMP and TIMP elevation. Co-administered HBO failed to attenuate these early deleterious effects, independent of functional improvement. CONCLUSIONS: The long-term consequences of simultaneously applied tPA and both NBO and HBO need to be addressed by further studies to identify therapeutic potencies in acute stroke, and to avoid unfavorable courses following combined treatment.