Brain Kynurenine Pathway and Functional Outcome of Rats Resuscitated From Cardiac Arrest

Background Brain injury and neurological deficit are consequences of cardiac arrest (CA), leading to high morbidity and mortality. Peripheral activation of the kynurenine pathway (KP), the main catabolic route of tryptophan metabolized at first into kynurenine, predicts poor neurological outcome in patients resuscitated after out‐of‐hospital CA. Here, we investigated KP activation in hippocampus and plasma of rats resuscitated from CA, evaluating the effect of KP modulation in preventing CA‐induced neurological deficit. Methods and Results Early KP activation was first demonstrated in 28 rats subjected to electrically induced CA followed by cardiopulmonary resuscitation. Hippocampal levels of the neuroactive metabolites kynurenine, 3‐hydroxy‐anthranilic acid, and kynurenic acid were higher 2 hours after CA, as in plasma. Further, 36 rats were randomized to receive the inhibitor of the first step of KP, 1‐methyl‐DL‐tryptophan, or vehicle, before CA. No differences were observed in hemodynamics and myocardial function. The CA‐induced KP activation, sustained up to 96 hours in hippocampus (and plasma) of vehicle‐treated rats, was counteracted by the inhibitor as indicated by lower hippocampal (and plasmatic) kynurenine/tryptophan ratio and kynurenine levels. 1‐Methyl‐DL‐tryptophan reduced the CA‐induced neurological deficits, with a significant correlation between the neurological score and the individual kynurenine levels, as well as the kynurenine/tryptophan ratio, in plasma and hippocampus. Conclusions These data demonstrate the CA‐induced lasting activation of the first step of the KP in hippocampus, showing that this activation was involved in the evolving neurological deficit. The degree of peripheral activation of KP may predict neurological function after CA.

BiP Heterozigosity Aggravates Pathological Deterioration in Experimental Amyotrophic Lateral Sclerosis

In the present study, we investigated the involvement of the chaperone protein BiP (also known as GRP78 or Hspa5), a master regulator of intracellular proteostasis, in two mouse models of neurodegenerative diseases: amyotrophic lateral sclerosis (ALS) and Parkinson’s disease (PD). To this end, we used mice bearing partial genetic deletion of the BiP gene (BiP+/− mice), which, for the ALS model, were crossed with mutant SOD1 (mSOD1) transgenic mice to generate mSOD1/BiP+/− double mutant mice. Our data revealed a more intense neurological decline in the double mutants, reflected in a greater deterioration of the neurological score and rotarod performance, with also a reduced animal survival, compared to mSOD1 transgenic mice. Such worsening was associated with higher microglial (labelled with Iba-1 immunostaining) and, to a lesser extent, astroglial (labelled with GFAP immunostaining) immunoreactivities found in the double mutants, but not with a higher loss of spinal motor neurons (labelled with Nissl staining) in the spinal cord. The morphological analysis of Iba-1 and GFAP-positive cells revealed a higher presence of activated cells, characterized by elevated cell body size and shorter processes, in double mutants compared to mSOD1 mice with normal BiP expression. In the case of the PD model, BiP+/− mice were unilaterally lesioned with the parkinsonian neurotoxin 6-hydroxydopamine (6-OHDA). In this case, however, we did not detect a greater susceptibility to damage in mutant mice, as the motor defects caused by 6-OHDA in the pole test and the cylinder rearing test, as well as the losses in tyrosine hydroxylase-containing neurons and the elevated glial reactivity (labelled with CD68 and GFAP immunostaining) detected in the substantia nigra were of similar magnitude in BiP+/− mice compared with wildtype animals. Therefore, our findings support the view that a dysregulation of the protein BiP may contribute to ALS pathogenesis. As BiP has been recently related to cannabinoid type-1 (CB1) receptor function, our work also opens the door to future studies on a possible link between BiP and the neuroprotective effects of cannabinoids that have been widely reported in this neuropathological context. In support of this possibility, preliminary data indicate that CB1 receptor levels are significantly reduced in mSOD1 mice having partial deletion of BiP gene.

Posterior-only debridement, internal fixation, and interbody fusion using titanium mesh in the surgical treatment of thoracolumbar tuberculosis with spinal epidural abscess: a minimum 5-year follow-up

Objective To investigate the clinical efficacy and feasibility of posterior-only debridement, internal fixation, and interbody fusion using titanium mesh in the surgical treatment of thoracolumbar tuberculosis (TB) with spinal epidural abscess. Methods From January 2008 to January 2014, a total of 45 patients (27 male and 18 female) were reviewed. The patients were diagnosed with thoracolumbar TB with spinal epidural abscess. The patients underwent posterior-only debridement, internal fixation, and interbody fusion using titanium mesh. Hence, we assessed the intraoperative and postoperative complications, disease recurrences, kyphosis deformity correction, and neurological improvement following the American Spinal Injury Association (ASIA). We used SPSS 22.0 for the statistical analyses. An independent Student’s t-test was used for the analysis of preoperative and postoperative continuous variables. The value of P (P < 0.05) was considered statistically significant. Results The mean age of patients was 37.76 ± 10.94 years (17–59 years). The mean follow-up time was 82.76 ± 12.56 months (60–128 months). The mean kyphosis Cobb angle preoperative was 29.36 ± 13.29° (5–55°) and postoperative was 3.58 ± 5.44° (− 6–13°), given the value of P (P < 0.001). According to the neurological score by the ASIA scale, there were 3 cases of grade B, 11 cases of grade C, 16 cases of grade D, and 15 cases of grade E preoperatively. The neurological score improved by 1 ~ 2 grades. All patients achieved pain relief and the VAS score significantly reduced at the last follow-up (P<0.05). While 1 patient had cerebrospinal fluid leakage, 1 had a neurological complication, 1 had delayed surgical wound healing, and 1 had a disease recurrence. No pseudoarthrosis or implant failure occurred in our patients. All patients achieved solid bone graft fusion. Conclusion For thoracolumbar TB patients with spinal epidural abscess, posterior-only debridement, internal fixation, and interbody fusion using titanium mesh are safe and effective surgical treatments.

A preclinical randomized multicenter trial of anti-IL-17A treatment for acute ischemic stroke

Multiple consensus statements have called for preclinical randomized controlled trials (pRCT) to improve translation in stroke research. Here, we investigated the efficacy of IL-17A neutralizing antibodies in a multicentric pRCT using a murine stroke model. C57/Bl.6 mice were subjected to transient middle cerebral artery occlusion (tMCAO). Mice were randomly allocated (1:1). Either anti-IL-17A (500 microgram) or isotype antibody (500 microgram) were administered 1 h after tMCAO. Primary analysis of infarct volumes was done by MRI after three days. Secondary analysis included mortality, neurological score, neutrophil infiltration and the impact of the gut microbiome on treatment effects. Out of 136 mice, 109 mice were included in the analysis. Mixed model analysis revealed that the IL-17A neutralization significantly reduced infarct sizes (anti IL-17A: 61.77 mm3 (SD: 31.04); IgG control: 75.66 mm3 (SD: 34.79); p=0.01). Secondary outcome measures showed a decrease in mortality (Hazard Ratio=3.43, 95% CI = 1.157 - 10.18; p=0.04) and neutrophil invasion into ischemic cortices. There was no difference in the neurological score. The analysis of the gut microbiome showed significant differences between centers. Taken together, this is the first positive pRCT in an ischemia reperfusion model. It suggests IL-17A neutralization as a potential target in stroke.

Network pharmacology and molecular docking reveal the effective substances and active mechanisms of Dalbergia Odoriferain protecting against ischemic stroke

Dalbergia Odorifera (DO) has been widely used for the treatment of cardiovascular and cerebrovascular diseasesinclinical. However, the effective substances and possible mechanisms of DO are still unclear. In this study, network pharmacology and molecular docking were used toelucidate the effective substances and active mechanisms of DO in treating ischemic stroke (IS). 544 DO-related targets from 29 bioactive components and 344 IS-related targets were collected, among them, 71 overlapping common targets were got. Enrichment analysis showed that 12 components were the possible bioactive components in DO, which regulating 9 important signaling pathways in 3 biological processes including ‘oxidative stress’ (KEGG:04151, KEGG:04068, KEGG:04915), ‘inflammatory response’(KEGG:04668, KEGG:04064) and ‘vascular endothelial function regulation’(KEGG:04066, KEGG:04370). Among these, 5 bioactive components with degree≥20 among the 12 potential bioactive components were selected to be docked with the top5 core targets using AutodockVina software. According to the results of molecular docking, the binding sites of core target protein AKT1 and MOL002974, MOL002975, and MOL002914 were 9, 8, and 6, respectively, and they contained 2, 1, and 0 threonine residues, respectively. And some binding sites were consistent, which may be the reason for the similarities and differences between the docking results of the 3 core bioactive components. The results of in vitro experiments showed that OGD/R could inhibit cell survival and AKT phosphorylation which were reversed by the 3 core bioactive components. Among them, MOL002974 (butein) had a slightly better effect. Therefore, the protective effect of MOL002974 (butein) against cerebral ischemia was further evaluated in a rat model of middle cerebral artery occlusion (MCAO) by detecting neurological score, cerebral infarction volume and lactate dehydrogenase (LDH) level. The results indicated that MOL002974 (butein) could significantly improve the neurological score of rats, decrease cerebral infarction volume, and inhibit the level of LDH in the cerebral tissue and serum in a dose-dependent manner. In conclusion, network pharmacology and molecular docking predicate the possible effective substances and mechanisms of DO in treating IS. And the results are verified by the in vitro and in vivo experiments. This research reveals the possible effective substances from DO and its active mechanisms for treating IS and provides a new direction for the secondary development of DO for treating IS.

Posterior-only Debridement, Internal Fixation, and Interbody Fusion Using Titanium Mesh in the Surgical Treatment of Thoracolumbar Tuberculosis With Spinal Epidural Abscess: A Minimum 5-year Follow-up

Objective: To investigate the clinical efficacy and feasibility of posterior-only debridement, internal fixation, and interbody fusion using titanium mesh in the surgical treatment of thoracolumbar tuberculosis (TB) with spinal epidural abscess.Methods: From January 2008 to January 2014, a total of 45 patients (27 male and 18 female) were reviewed. The patients were diagnosed with thoracolumbar TB with spinal epidural abscess. The patients underwent posterior-only debridement, internal fixation, and interbody fusion using titanium mesh. Hence, we assessed the intraoperative and postoperative complications, disease recurrences, kyphosis deformity correction, and neurological improvement following the American Spinal Injury Association (ASIA). We used SPSS 22.0 for the statistical analyses. An independent Student’s t-test was used for the analysis of preoperative and postoperative continuous variables. The value of P (P<0.05) was considered statistically significant.Results: The mean age of patients was 37.76 ±10.94 years (17–59 years). The mean follow-up time was 82.76 ±12.56 months (60–128 months). The mean kyphosis Cobb angle preoperative was 29.36 ±13.29° (5–55°) and postoperative was 3.58 ±5.44° (–6–13°), given the value of P (P<0.001). According to the neurological score by the ASIA scale, there were 3 cases of grade B, 11 cases of grade C, 16 cases of grade D, and 15 cases of grade E preoperatively. The neurological score improved by 1~2 grades. All patients achieved pain relief and the VAS score significantly reduced at the last follow-up (P＜0.05). While 1 patient had cerebrospinal fluid leakage, 1 had a neurological complication, 1 had delayed surgical wound healing, and 1 had a disease recurrence. No pseudoarthrosis or implant failure occurred in our patients. All patients achieved solid bone graft fusion.Conclusion: For thoracolumbar TB patients with spinal epidural abscess, posterior-only debridement, internal fixation, and interbody fusion using titanium mesh are safe and effective surgical treatments.*As a note, Qile Gao and Chaofei Han are Co-First authors.

Baicalein Mediates Mitochondrial Autophagy via miR-30b and the NIX/BNIP3 Signaling Pathway in Parkinson’s Disease

Parkinson’s disease (PD) is regarded as a severe neurodegenerative disorder. Baicalein is involved in the treatment of PD. This study explored the mechanism of baicalein in PD. The PD rat model was established using 6-hydroxydopamine. The neurologic score, dopamine (DA) content, apoptotic cells, and neuronal damage were evaluated after rats were treated with baicalein. Autophagy in PD rats was inhibited using 3-methyladenine (3-MA). The mitochondrial membrane potential (MMP) and autophagy-related proteins (LC3, P62) were detected. Next, agomiR-30b was transfected into PD rats. The targeting relation between miR-30b and NIX was predicted and verified. Then, sh-NIX was transfected into PD rats, and the effects of miR-30b and NIX on MMP, LC3, and P62 were assessed. When miR-30b was overexpressed using agomiR-30b, the NIX and BNIP3 levels were detected. Baicalein increased the neurological score and restored DA content, neurons, MMP, and mitochondrial autophagy protein levels. Baicalein inhibited miR-30b expression and miR-30b targeted NIX. miR-30b upregulation or NIX silencing reversed the effect of baicalein on MMP and mitochondrial autophagy. Baicalein upregulated NIX and BNIP3 expressions, while miR-30b overexpression inhibited NIX and BNIP3 expressions. In summary, baicalein mediated mitochondrial autophagy and restored neuronal activity by downregulating miR-30b and activating the NIX/BNIP3 pathway, thus protecting against PD.

Metformin prevents stroke damage in non-diabetic female mice with chronic kidney disease

Chronic kidney disease (CKD) worsens ischemic stroke severity in both patients and animals. In mice, these poorer functional outcomes are associated with decreased brain activity of AMP-activated protein kinase (AMPK), a molecule that recently emerged as a potential therapeutic target for ischemic stroke. The antidiabetic drug metformin, a well-known activator of AMPK, has improved stroke outcomes in diabetic patients with normal renal function. We investigated whether chronic metformin pre-conditioning can rescue AMPK activity and prevent stroke damage in non-diabetic mice with CKD. Eight-week-old female C57BL/6J mice were assigned to CKD or SHAM groups. CKD was induced through right kidney cortical electrocautery, followed by left total nephrectomy. Mice were then allocated to receive metformin (200 mg/kg/day) or vehicle for 5 weeks until stroke induction by transient middle cerebral artery occlusion (tMCAO). The infarct volumes were lower in CKD mice exposed to metformin than in vehicle-treated CKD mice 24 h after tMCAO. Metformin pre-conditioning of CKD mice improved their neurological score, grip strength, and prehensile abilities. It also enhanced AMPK activation, reduced apoptosis, increased neuron survival and decreased microglia/macrophage M1 signature gene expression as well as CKD-induced activation of the canonical NF-κB pathway in the ischemic lesions of CKD mice.

Morphological characteristic of intracerebral hemorrhage in rats and correlation of its volume with results of behavioral tests

The intracerebral hemorrhage is associated with severe complications and high mortality. Currently there are no effective methods of treatment of this disease while the standard collagenase model of intracerebral hemorrhage is not described sufficiently. Objective. To analyze morphological characteristics of the collagenase model of intracerebral hemorrhage, and develop the regression formula predicting the hemorrhage volume based on the results of behavioral tests. Materials and methods. The experiments were carried out on 7 white male rats weighing 250-400 g aged 11-13 months. All animals underwent surgery to simulate intracerebral hemorrhage. Rats were anesthetized and then, stereotactically, using a needle with a diameter of 0.47 mm, 0.2 units of collagenase type IV were slowly injected into the left striatum. The day after the intracerebral hemorrhage, functional disabilities developed in rats were studied using beam walking test, neurological score test and adhesive removal test. Immediately after performing behavioral tests, the rats were sacrificed by decapitation. After the brain formalin fixation, serial sections on a vibromicrotome of 200 µm thick each in the anteroposterior direction with following morphological examination were made. Results. It was revealed that the collagenase model of intracerebral hemorrhage is associated with a large variability of the hemorrhage volume. It also had an irregular rugged shape and marks of repeated diapedetic hemorrhages of about 0.6 mm depth. The center of intracerebral hemorrhage along the anteroposterior axis was in average 0.5 mm posterior of the actual site of collagenase injection.The combined use of the neurological score test, the beam walking test and the adhesive removal test in the collagenase model can help estimate the probable intracerebral hemorrhage volume on the 1st day using the regression formula. Conclusions. Technical details identified in our study can help researchers in planning and conduction of correct experiments related to intracerebral hemorrhage.

Abstract P422: Chronic Nicotine Exposure Increases Hematoma Growth Following Intracerebral Hemorrhage in Young Rats of Both Sexes

Spontaneous intracerebral hemorrhage (sICH) is the deadliest stroke subtype. There is strong evidence that tobacco use / smoking increases the risk of sICH, and some epidemiological studies have observed sex differences in sICH outcomes. However, systematic controlled studies on the effect of tobacco / smoking on post-sICH outcomes in both sexes are lacking. Therefore, we determined the effect of nicotine exposure on outcomes following collagenase-induced sICH both sexes. Young animals of both sexes were randomly divided into nicotine (4.5 mg / kg / day b.w.) and vehicle (saline) treatment groups (using osmotic pumps for two to three weeks). sICH in females was induced during the diestrous stage of estrous cycle. sICH was induced by collagenase injection into the right striatum and ~24 hours later, neurological scores were evaluated, rats were euthanized, and brains were sectioned to measure hematoma volume. Hematoma volumes for male rats was 42% higher (p<0.01) in the nicotine-treated group (139 ± 9 mm 3 , n=10) versus vehicle-treated group (98 ± 9 mm 3 , n=10). Hematoma volumes for female rats was 48% higher (p<0.01) in the nicotine-treated group (134 ± 11 mm 3 , n=10) versus vehicle-treated group (90 ± 7 mm 3 , n=10). Hematoma volumes for the vehicle and nicotine-treated male groups were not different from their respective female groups. The neurological score for the nicotine-treated male group (9.3 ± 0.6) was significantly higher (p<0.05) when compared to vehicle group (7.4 ± 0.6). The neurological score for the nicotine-treated female group (10.7 ± 0.2) was significantly higher (p<0.001) than the vehicle group (7.7 ± 0.7). The neurological score for the vehicle-treated male group was not different from its respective female group. However, the neurological score for the nicotine-treated male group was significantly lower than the female group. Our results show that chronic nicotine exposure increases hematoma volume post-sICH in animals of both sexes. Future studies into the mechanism of nicotine-induced increase in hematoma growth following sICH are required. Support: James and Esther King Biomedical Research Grant 9JK08