Rodent Models Of Injury Research Articles

Contusion Rodent Model of Traumatic Brain Injury: Controlled Cortical Impact.

Traumatic brain injury (TBI) is a heterogeneous brain injury which represents one of the leading causes of mortality and disability worldwide. Rodent TBI models are helpful to examine the cellular and molecular mechanisms after injury. Controlled cortical impact (CCI) is one of the most commonly used TBI models in rats and mice, based on its consistency of injury and ease of implementation. Here, we describe a CCI protocol to induce a moderate contusion to the somatosensory motor cortex. We provide additional protocols for monitoring animals after CCI induction.

Repetitive Erythropoietin Treatment Improves Long-Term Neurocognitive Outcome by Attenuating Hyperoxia-Induced Hypomyelination in the Developing Brain.

Introduction: Preterm infants born before 28 weeks of gestation are at high risk of neurodevelopmental impairment in later life. Cerebral white and gray matter injury is associated with adverse outcomes. High oxygen levels, often unavoidable in neonatal intensive care, have been identified as one of the main contributing factors to preterm brain injury. Thus, preventive and therapeutic strategies against hyperoxia-induced brain injury are needed. Erythropoietin (Epo) is a promising and also neuroprotective candidate due to its clinical use in infants as erythropoiesis-stimulating agent.Objective: The objective of this study was to investigate the effects of repetitive Epo treatment on the cerebral white matter and long-term motor-cognitive outcome in a neonatal rodent model of hyperoxia-induced brain injury.Methods: Three-day old Wistar rats were exposed to hyperoxia (48 h, 80% oxygen). Four doses of Epo (5,000 IU/kg body weight per day) were applied intraperitoneally from P3-P6 with the first dose at the onset of hyperoxia. Oligodendrocyte maturation and myelination were evaluated via immunohistochemistry and Western blot on P11. Motor-cognitive deficits were assessed in a battery of complex behavior tests (Open Field, Novel Object Recognition, Barnes maze) in adolescent and fully adult animals. Following behavior tests animals underwent post-mortem diffusion tensor imaging to investigate long-lasting microstructural alterations of the white matter.Results: Repetitive treatment with Epo significantly improved myelination deficits following neonatal hyperoxia at P11. Behavioral testing revealed attenuated hyperoxia-induced cognitive deficits in Epo-treated adolescent and adult rats.Conclusion: A multiple Epo dosage regimen protects the developing brain against hyperoxia-induced brain injury by improving myelination and long-term cognitive outcome. Though current clinical studies on short-term outcome of Epo-treated prematurely born children contradict our findings, long-term effects up to adulthood are still lacking. Our data support the essential need for long-term follow-up of preterm infants in current clinical trials.

Significance of Omega-3 Fatty Acids in the Prophylaxis and Treatment after Spinal Cord Injury in Rodent Models.

Polyunsaturated fatty acids (ω-3 acids, PUFAs) are essential components of cell membranes in all mammals. A multifactorial beneficial influence of ω-3 fatty acids on the health of humans and other mammals has been observed for many years. Therefore, ω-3 fatty acids and their function in the prophylaxis and treatment of various pathologies have been subjected to numerous studies. Regarding the documented therapeutic influence of ω-3 fatty acids on the nervous and immune systems, the aim of this paper is to present the current state of knowledge and the critical assessment of the role of ω-3 fatty acids in the prophylaxis and treatment of spinal cord injury (SCI) in rodent models. The prophylactic properties (pre-SCI) include the stabilization of neuron cell membranes, the reduction of the expression of inflammatory cytokines (IL-1β, TNF-α, IL-6, and KC/GRO/CINC), the improvement of local blood flow, reduced eicosanoid production, activation of protective intracellular transcription pathways (dependent on RXR, PPAR-α, Akt, and CREB), and increased concentration of lipids, glycogen, and oligosaccharides by neurons. On the other hand, the therapeutic properties (post-SCI) include the increased production of endogenous antioxidants such as carnosine and homocarnosine, the maintenance of elevated GSH concentrations at the site of injury, reduced concentrations of oxidative stress marker (MDA), autophagy improvement (via increasing the expression of LC3-II), and p38 MAPK expression reduction in the superficial dorsal horns (limiting the sensation of neuropathic pain). Paradoxically, despite the well-documented protective activity of ω-3 acids in rodents with SCI, the research does not offer an answer to the principal question of the optimal dose and treatment duration. Therefore, it is worth emphasizing the role of multicenter rodent studies with the implementation of standards which initially may even be based on arbitrary criteria. Additionally, basing on available research data, the authors of this paper make a careful attempt at referring some of the conclusions to the human population.

Glucagon-like peptide-1 receptor mediates the beneficial effect of liraglutide in an acute lung injury mouse model involving the thioredoxin-interacting protein.

Repurposing clinically used drugs is among the important strategies in drug discovery. Glucagon-like peptide-1 (GLP-1) and its diabetes-based drugs, such as liraglutide, possess a spectrum of extra-pancreatic functions, while GLP-1 receptor (GLP-1R) is most abundantly expressed in the lung. Recent studies have suggested that GLP-1-based drugs exert beneficial effects in chronic, as well as acute, lung injury rodent models. Here, we show that liraglutide pretreatment reduced LPS induced acute lung injury in mice. It significantly reduced lung injury score, wet/dry lung weight ratio, bronchoalveolar lavage fluid immune cell count and protein concentration, and cell apoptosis in the lung, and it was associated with reduced lung inflammatory cytokine and chemokine gene expression. Importantly, these effects were virtually absent in GLP-1R−/− mice. A well-known function of GLP-1 and GLP-based drugs in pancreatic β-cells is the attenuation of high-glucose stimulated expression of thioredoxin-interacting protein (TxNIP), a key component of inflammasome. LPS-challenged lungs showed elevated TxNIP mRNA and protein expression, which was attenuated by liraglutide treatment in a GLP-1R-dependent manner. Hence, our observations suggest that GLP-1R is essential in mediating beneficial effects of liraglutide in acute lung injury, with the inflammasome component TxNIP as a potential target.

Subacute metformin treatment reduces inflammation and improves functional outcome following neonatal hypoxia ischemia

Hypoxia-ischemia (HI) injury is a leading cause of neonatal death and long-term disability, and existing treatment options for HI offer only modest benefit. Early intervention with the drug metformin has been shown to promote functional improvement in numerous rodent models of injury and has pleiotropic cellular effects in the brain. We have previously shown that 1 week of metformin treatment initiated 24 ​h after HI in neonatal mice resulted in improved motor and cognitive performance, activation of endogenous neural precursor cells (NPCs), and increased oligodendrogenesis. While promising, a limitation to this work is that immediate pharmacological intervention is not always possible in the clinic. Herein, we investigated whether delaying metformin treatment to begin in the subacute phase post-HI would still effectively promote recovery. Male and female C57/BL6 mice received HI injury postnatally, and metformin treatment began 7 days post-HI for up to 4 weeks. Motor and cognitive performance was assessed across time using behavioural tests (cylinder, foot fault, puzzle box). We found that metformin improved motor and cognitive behaviour, decreased inflammation, and increased oligodendrocytes in the motor cortex. Our present findings demonstrate that a clinically relevant subacute metformin treatment paradigm affords the potential to treat neonatal HI, and that improved outcomes occur through modulation of the inflammatory response and oligodendrogenesis.

European Society for Sexual Medicine Consensus Statement on the Use of the Cavernous Nerve Injury Rodent Model to Study Postradical Prostatectomy Erectile Dysfunction.

IntroductionRodent animal models are currently the most used in vivo model in translational studies looking into the pathophysiology of erectile dysfunction after nerve-sparing radical prostatectomy.AimThis European Society for Sexual Medicine (ESSM) statement aims to guide scientists toward utilization of the rodent model in an appropriate, timely, and proficient fashion.MethodsMEDLINE and EMBASE databases were searched for basic science studies, using a rodent animal model, looking into the consequence of pelvic nerve injury on erectile function.Main outcome measuresThe authors present a consensus on how to best perform experiments with this rodent model, the details of the technique, and highlight possible pitfalls.ResultsOwing to the specific issue—basic science—Oxford 2011 Levels of Evidence criteria cannot be applied. However, ESSM statements on this topic will be provided in which we summarize the ESSM position on various aspects of the model such as the use of the Animal Research Reporting In Vivo Experiments guideline and the of common range parameter for nerve stimulation. We also highlighted the translational limits of the model.ConclusionThe following statements were formulated as a suggestive guidance for scientists using the cavernous nerve injury model. With this, we hope to standardize and further improve the quality of research in this field. It must be noted that this model has its limitations.Weyne E, Ilg MM, Cakir OO, et al. European Society for Sexual Medicine Consensus Statement on the Use of the Cavernous Nerve Injury Rodent Model to Study Postradical Prostatectomy Erectile Dysfunction. Sex Med 2020;8:327–337.

Sacubitril/Valsartan Improves Cardiac Function and Decreases Myocardial Fibrosis Via Downregulation of Exosomal miR-181a in a Rodent Chronic Myocardial Infarction Model.

BackgroundExosomes are small extracellular vesicles that function as intercellular messengers and effectors. Exosomal cargo contains regulatory small molecules, including miRNAs, mRNAs, lncRNAs, and small peptides that can be modulated by different pathological stimuli to the cells. One of the main mechanisms of action of drug therapy may be the altered production and/or content of the exosomes.Methods and ResultsWe studied the effects on exosome production and content by neprilysin inhibitor/angiotensin receptor blockers, sacubitril/valsartan and valsartan alone, using human‐induced pluripotent stem cell‐derived cardiomyocytes under normoxic and hypoxic injury model in vitro, and assessed for physiologic correlation using an ischemic myocardial injury rodent model in vivo. We demonstrated that the treatment with sacubitril/valsartan and valsartan alone resulted in the increased production of exosomes by induced pluripotent stem cell‐derived cardiomyocytes in vitro in both conditions as well as in the rat plasma in vivo. Next‐generation sequencing of these exosomes exhibited downregulation of the expression of rno‐miR‐181a in the sacubitril/valsartan treatment group. In vivo studies employing chronic rodent myocardial injury model demonstrated that miR‐181a antagomir has a beneficial effect on cardiac function. Subsequently, immunohistochemical and molecular studies suggested that the downregulation of miR‐181a resulted in the attenuation of myocardial fibrosis and hypertrophy, restoring the injured rodent heart after myocardial infarction.ConclusionsWe demonstrate that an additional mechanism of action of the pleiotropic effects of sacubitril/valsartan may be mediated by the modulation of the miRNA expression level in the exosome payload.

Nogo receptor decoy promotes recovery and corticospinal growth in non-human primate spinal cord injury.

After CNS trauma such as spinal cord injury, the ability of surviving neural elements to sprout axons, reorganize neural networks and support recovery of function is severely restricted, contributing to chronic neurological deficits. Among limitations on neural recovery are myelin-associated inhibitors functioning as ligands for neuronal Nogo receptor 1 (NgR1). A soluble decoy (NgR1-Fc, AXER-204) blocks these ligands and provides a means to promote recovery of function in multiple preclinical rodent models of spinal cord injury. However, the safety and efficacy of this reagent in non-human primate spinal cord injury and its toxicological profile have not been described. Here, we provide evidence that chronic intrathecal and intravenous administration of NgR1-Fc to cynomolgus monkey and to rat are without evident toxicity at doses of 20 mg and greater every other day (≥2.0 mg/kg/day), and far greater than the projected human dose. Adult female African green monkeys underwent right C5/6 lateral hemisection with evidence of persistent disuse of the right forelimb during feeding and right hindlimb during locomotion. At 1 month post-injury, the animals were randomized to treatment with vehicle (n = 6) or 0.10-0.17 mg/kg/day of NgR1-Fc (n = 8) delivered via intrathecal lumbar catheter and osmotic minipump for 4 months. One animal was removed from the study because of surgical complications of the catheter, but no treatment-related adverse events were noted in either group. Animal behaviour was evaluated at 6-7 months post-injury, i.e. 1-2 months after treatment cessation. The use of the impaired forelimb during spontaneous feeding and the impaired hindlimb during locomotion were both significantly greater in the treatment group. Tissue collected at 7-12 months post-injury showed no significant differences in lesion size, fibrotic scar, gliosis or neuroinflammation between groups. Serotoninergic raphespinal fibres below the lesion showed no deficit, with equal density on the lesioned and intact side below the level of the injury in both groups. Corticospinal axons traced from biotin-dextran-amine injections in the left motor cortex were equally labelled across groups and reduced caudal to the injury. The NgR1-Fc group tissue exhibited a significant 2-3-fold increased corticospinal axon density in the cervical cord below the level of the injury relative to the vehicle group. The data show that NgR1-Fc does not have preclinical toxicological issues in healthy animals or safety concerns in spinal cord injury animals. Thus, it presents as a potential therapeutic for spinal cord injury with evidence for behavioural improvement and growth of injured pathways in non-human primate spinal cord injury.

Tissue-Engineered Human Myobundle System as a Platform for Evaluation of Skeletal Muscle Injury Biomarkers.

Traditional serum biomarkers used to assess skeletal muscle damage, such as activity of creatine kinase (CK), lack tissue specificity and sensitivity, hindering early detection of drug-induced myopathies. Recently, a novel four-factor skeletal muscle injury panel (MIP) of biomarkers consisting of skeletal troponin I (sTnI), CK mass (CKm), fatty-acid-binding protein 3 (Fabp3), and myosin light chain 3, has been shown to have increased tissue specificity and sensitivity in rodent models of skeletal muscle injury. Here, we evaluated if a previously established model of tissue-engineered functional human skeletal muscle (myobundle) can allow detection of the MIP biomarkers after injury or drug-induced myotoxicity in vitro. We found that concentrations of three MIP biomarkers (sTnI, CKm, and Fabp3) in myobundle culture media significantly increased in response to injury by a known snake venom (notexin). Cerivastatin, a known myotoxic statin, but not pravastatin, induced significant loss of myobundle contractile function, myotube atrophy, and increased release of both traditional and novel biomarkers. In contrast, dexamethasone induced significant loss of myobundle contractile function and myotube atrophy, but decreased the release of both traditional and novel biomarkers. Dexamethasone also increased levels of matrix metalloproteinase-2 and -3 in the culture media which correlated with increased remodeling of myobundle extracellular matrix. In conclusion, this proof-of-concept study demonstrates that tissue-engineered human myobundles can provide an in vitro platform to probe patient-specific drug-induced myotoxicity and performance assessment of novel injury biomarkers to guide preclinical and clinical drug development studies.

Opposite effects of the FXR agonist obeticholic acid on Mafg and Nrf2 mediate the development of acute liver injury in rodent models of cholestasis

The farnesoid-X-receptor (FXR) is validated target in the cholestatic disorders treatment. Obeticholic acid (OCA), the first in class of FXR agonist approved for clinical use, causes side effects including acute liver decompensation when administered to cirrhotic patients with primary biliary cholangitis at higher than recommended doses. The V-Maf avian-musculoaponeurotic-fibrosarcoma-oncogene-homolog-G (Mafg) and nuclear factor-erythroid-2-related-factor-2 (Nrf2) mediates some of the downstream effects of FXR. In the present study we have investigated the role of FXR/MafG/NRF2 pathway in the development of liver toxicity caused by OCA in rodent models of cholestasis. Cholestasis was induced by bile duct ligation (BDL) or administration of α-naphtyl-isothiocyanate (ANIT) to male Wistar rats and FXR−/− and FXR+/+ mice. Treating BDL and ANIT rats with OCA exacerbated the severity of cholestasis, hepatocytes injury and severely downregulated the expression of basolateral transporters. In mice, genetic ablation FXR or its pharmacological inhibition by 3-(naphthalen-2-yl)-5-(piperidin-4-yl)-1,2,4-oxadiazole rescued from negative regulation of MRP4 and protected against liver injury caused by ANIT. By RNAseq analysis we found that FXR antagonism effectively reversed the transcription of over 2100 genes modulated by OCA/ANIT treatment, including Mafg and Nrf2 and their target genes Cyp7a1, Cyp8b1, Mat1a, Mat2a, Gss. Genetic and pharmacological Mafg inhibition by liver delivery of siRNA antisense or S-adenosylmethionine effectively rescued from damage caused by ANIT/OCA. In contrast, Nrf2 induction by sulforaphane was protective. ConclusionsLiver injury caused by FXR agonism in cholestasis is FXR-dependent and is reversed by FXR and Mafg antagonism or Nrf2 induction.

Signaling Mechanisms in Cognition

Signaling in the gastrointestinal tract is of great interest as a contributing factor to the development of cognition. The dysregulation of Gut‐Brain‐Axis (GBA) signaling may be associated with gastrointestinal manifestations preceding impairments in the cognitive and/or memory and learning process, supporting the hypothesis that it starts from the gut and spreads to the brain ( Braak et al. 2013). The overall goal of this project is to identify how the gut signals to the brain via gut‐synthesized molecules in relation to learning and memory. We suggest that α‐synuclein in the gut plays a key role in gut injury in response to lipopolysaccharide (LPS) and alcohol. Its aggregation possibly initiates the spreading of toxicity from the gut to the brain via the vagus nerve. This could cause an increase in hyper‐phosphorylated tau protein and repression of protective BDNF, leading to cognitive impairment. There are several highly innovative aspects of our proposal. Preliminary data showed increased α‐synuclein in LPS/alcohol injury in rodent models. Here, we propose to use a more sophisticated model: Wild Type (WT) and SNCA knockout (KO) mice treated and non‐treated with LPS/alcohol. Specifically, we will assess whether alterations of the intestinal barrier (or leaky guts) enhances damage as well as the increased production of α‐synuclein. The cognitive function test has been successfully performed using the Morris Water Maze (MWM) in murine model (APP‐mice). Since the mechanisms of GBA are still being worked out, the proposed objectives were to assess the time course and thereby threshold of the compromised intestinal barrier capable of influencing brain function, specifically cognitive and memory function in mice challenged with gut injury. Given the intense bidirectional interaction of the gut with the brain influencing neuronal activity, behavior (cognitive and memory), as well as levels of neurotrophic factors and inflammatory processes, the proposed studies may reshape our understanding of the etiology of cognitive and memory functions.

Ischemia‐induced vascular congestion of the ascending vasa recta precedes congestion of the peritubular capillaries in the renal medulla

Vascular congestion, or the aggregation of red blood cells in the microvasculature of the renal medulla is a prominent finding in ischemic acute kidney injury (AKI). Studies in rodent models of ischemia‐reperfusion injury (IRI) have suggested that vascular congestion occurs during the ischemic period and worsens following reperfusion, however, the location(s) in which congestion originates during ischemia have not been defined. We hypothesized that ‘vascular congestion first occurs in the medullary vasa recta (VR) capillaries during the ischemic period, prior to congestion of peritubular (PT) capillaries during reperfusion’. To test this hypothesis, a 45‐minute warm bilateral ischemia was performed in male WKY rats (10 wks old). Rats were randomized to 0, 1, 2, 6, 10, or 24 hour(s) of reperfusion (n=5/group). Congestion of the cortical PT capillaries, medullary VR and PT capillaries, and inner medulla (IM) was scored (blinded; scale: 0–5, 0=0% congestion, 5=100% congestion) in histological sections. To localize congestion within the VR, immunofluorescence of neural‐glial antigen 2 (NG2) was assessed. Medullary VR congestion score was 4.5 (90%) at time 0 (no reperfusion) and remained elevated at 1–10 hours of reperfusion. At 24 hours of reperfusion, VR congestion had significantly decreased to 30% (pANOVA= 0.004, post hoc p=0.002). Medullary PT congestion score was 0.5 (10%) at time 0 and was significantly less than the VR congestion at time 0 (90%) (pINTERACTION=0.004, post hoc p<0.0001). Non‐quantitative analysis of NG2 staining, localized congestion to the ascending VR. At time 0, IM congestion score was 0.5 (10%). IM congestion increased to 4 (80%) by 2 hours of reperfusion (pANOVA=0.022, post hoc p=0.019) and remained elevated through 10 hours of reperfusion, however by 24 hours of reperfusion IM congestion had decreased. Congestion of the cortical PT capillary congestion was significantly greater at time 0 and had an average score of 2.5 (50%) (pANOVA=0.0005). Following 1, 2, or 6 hour reperfusion, cortical PT congestion decreased to less than 10%, and with 10 or 24 hour reperfusion there was no cortical congestion. It has been reported that congestion occurs during ischemia and increases following reperfusion, however the localization of medullary congestion over the initial 24 hours of reperfusion remains unknown. Importantly, our data provides the first evidence that medullary congestion originates in the VR capillaries during the ischemic period and is evident prior to congestion in the PT capillaries during reperfusion. Pericytes are contractile cells which surround the descending vasa recta (DVR). Using a pericyte marker, NG2, we found that congestion was not in NG2‐positive DVR but was localized to the ascending VR (NG2‐negative). Vascular congestion of the renal medulla has been reported to be the primary factor contributing to non‐recovery from ischemic AKI. Understanding how, and where, congestion occurs in the renal medulla is critical to find therapeutic targets for congestion in order to improve recovery from ischemic AKI. We speculate that medullary congestion of AVR may drive blood into the PT capillaries during reperfusion where it then becomes trapped which may prolong ischemia and worsen injury.Support or Funding InformationNIH PO1HL134604 and DK099548

Microbiome Signatures Associated with Rodent Model of Traumatic Brain Injury vs. Psychological Stress

Traumatic brain injury (TBI) and post‐traumatic stress disorder (PTSD) are two signature illnesses of modern warfare. Discrimination between psychological stress and TBI using a knowledge‐driven unbiased panel of biomarker signatures will be essential for designing precise care management. Recent data suggest a role of alterations of the gut micobiome architecture susceptible to both brain injury and psychological disorders. The present study was conducted to identify factors discriminating between psychological stress and TBI in the fecal microbiome of rats and further to study the effect of diets enriched with varied polyunsaturated fat compositions.A closed‐head TBI model consisting of blast overpressure (BOP) wave exposure coupled with a weight drop concussion (Marmarou method) was used on a group of adult male rats (N=12 each exposed and sham). In parallel, an independent group of rats (N=12 each exposed and sham) was subjected to an underwater trauma (UWT) stressor model that consisted of 30s of swimming and habituation, followed by 30s of forced whole body immersion. Prior to the stress types (BOP or UWT), animals were maintained for six weeks and continued thereafter on two different diets (N=4 per diet group): Standard house chow and custom chow. Unlike the house chow, the custom diets contained no long chain ω‐3 polyunsaturated fatty acids to offset their high Linoleic acid content. The fecal bacterial populations were characterized by identification of 16S ribosomal RNA.Principal coordinate analysis showed clear separation between taxonomic phylogenetic profiles linked to the stress types from their corresponding shams. Within each stress type, the rodents fed on house chow showed different fecal microbial population from the customized diet. Firmicutes, Proteobacteria, and Cyanobacteria were those phyla of interest that showed significant population variance caused by the stress and diet variants. We integrated this microbial profile with rodent blood and brain genomic profile assayed in parallel. The result suggested a significant contribution of gut‐microbiome in energy networks related to the stress response, and diet emerged as a strong candidate in regulating resilience. This outcome could lead in identifying novel customized treatment of TBI and psychological stress.Support or Funding InformationThe project was funded by DoD‐MOMRPThe differential impact of two neurological stress models on different component of gut‐brain axisFigure 1

3β‐Diol Attenuates COX‐2 Levels in a Neonatal In‐Vivo and In‐Vitro Ischemic Injury Rodent and Human Model

Studies using experimental stroke models demonstrate that neuroprotective agents which provide protection from secondary injury, including inflammation, result in improved outcome. Estrogen receptor β (ESR2) has the potential to be a novel therapeutic target in the treatment of ischemic stroke. Previous studies have shown that ESR2 activation elicits beneficial effects in the cerebrovasculature by promoting vasorelaxation and by attenuating pro‐inflammatory mediator production. We previously observed that activation of the androgen, 5α‐androstane‐3β,17β‐diol (3β‐diol), reduced pro‐inflammatory mediator production in adult human vascular smooth muscle cells and rodent cerebral arteries, and this response was ESR2 dependent. The current study was designed to determine if ERS2 is expressed in both a human pediatric brain endothelial cell model and juvenile rat stroke model and whether 3β‐diol will attenuate cyclooxygenase‐2 (COX‐2) expression following ischemic injury. Male rat pups (P10–15) underwent 15 minutes of bilateral internal carotid artery occlusion (BCAO) or sham surgery and 24‐hour reperfusion. At the onset of reperfusion, pups were injected with 3β‐diol i.p. (2.5mg/kg) or vehicle (1.2% EtOH and 98.8% PEG). Next, animals were heavily sedated and whole brain removed at 24 hours. In an in‐vitro model of ischemic stroke, male primary pediatric human microvascular endothelial cells (HBMEC; Cell Systems) were exposed to hypoxia plus glucose deprivation (HGD; 1% O2) or normoxia (21% O2) plus or minus 3β‐diol (10nM) or vehicle (0.0001% ethanol) for 3h at passage 7, using a humidified chamber (BioSpherix®) housed within a 5% CO2 incubator. Total protein was extracted from rat whole brain and human endothelial cells, assayed for protein content, and COX‐2 levels assessed using western analysis. RT‐PCR analysis was used to explore whether 3β‐diol alters expression of its target receptor, ERS2. We observed that ESR2 was basally expressed in the prepubescent brain and HBMECs, and expression was not altered following ischemic injury. Proinflammatory COX‐2 protein levels were increased in both whole brain and HBMEC homogenates from BCAO and HGD groups compared to sham and vehicle controls, and the selective ERS2 agonist, 3β‐diol, attenuated this response. In conclusion, these studies suggest that 3β‐diol may have a role in modulating ischemia‐induced COX‐2 during stroke. Since ERS2 is expressed in the whole brain and brain endothelial cells, and ERS2 levels are not decreased during ischemic injury, ERS2 may be a viable target for future stroke therapeutics in the pediatric population by attenuating inflammatory modulators during ischemic injury in the developing brain.Support or Funding InformationUniversity of Arizona Valley Research Partnership Grant VRP04 P1 (RJG), American Heart Association 19AIREA34480018 (RJG).

Common transcriptional signatures of neuropathic pain.

The dorsal root ganglia (DRG) are key structures in nociception and chronic pain disorders. Several gene expression studies of DRG in preclinical pain models have been performed, but it is unclear if consistent gene changes are identifiable. We, therefore, compared several recent RNA-Seq data sets on the whole DRG in rodent models of nerve injury. Contrary to previous findings, we show hundreds of common differentially expressed genes and high positive correlation between studies, despite model and species differences. We also find, in contrast to previous studies, that 60% of the common rodent gene response after injury is likely to occur in nociceptors of the DRG. Substantial expression changes are observed at a 1-week time-point, with smaller changes in the same genes at a later 3- to 4-week time-point. However, a subset of genes shows a similar magnitude of changes at both early and late time-points, suggesting their potential involvement in the maintenance of chronic pain. These genes are centred around suppression of endogenous opioid signalling. Reversal of this suppression could allow endogenous and exogenous opioids to exert their analgesic functions and may be an important strategy for treating chronic pain disorders. Currently used drugs, such as amitriptyline and duloxetine, do not seem to appropriately modulate many of the critical pain genes and indeed may transcriptionally suppress endogenous opioid signalling further.

3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury.

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of sciatic nerve injury rodent models. In this protocol, we describe a novel kinematic analysis method that uses a three-dimensional (3D) motion capture apparatus for functional evaluations using a rat sciatic nerve crush injury model. First, the rat is familiarized with treadmill walking. Markers are then attached to the designated bone landmarks and the rat is made to walk on the treadmill at the desired speed. Meanwhile, the posterior limb movements of the rat are recorded using four cameras. Depending on the software used, marker tracings are created using both automatic and manual modes and the desired data are produced after subtle adjustments. This method of kinematic analysis, which uses a 3D motion capture apparatus, offers numerous advantages, including superior precision and accuracy. Many more parameters can be investigated during the comprehensive functional evaluations. This method has several shortcomings that require consideration: The system is expensive, can be complicated to operate, and may produce data deviations due to skin shifting. Nevertheless, kinematic analysis using a 3D motion capture apparatus is useful for performing functional anterior and posterior limb evaluations. In the future, this method may become increasingly useful for generating accurate assessments of various traumas and diseases.

Molecular Dynamics of Lipopolysaccharide-Induced Lung Injury in Rodents.

Acute respiratory distress syndrome (ARDS) is a common disease entity in critical care medicine and is still associated with a high mortality. Because of the heterogeneous character of ARDS, animal models are an insturment to study pathology in relatively standardized conditions. Rodent models can bridge the gap from in vitro investigations to large animal and clinical trials by facilitating large sample sizes under physiological conditions at comparatively low costs. One of the most commonly used rodent models of acute lung inflammation and ARDS is administration of lipopolysaccharide (LPS), either into the airways (direct, pulmonary insult) or systemically (indirect, extra-pulmonary insult). This narrative review discusses the dynamics of important pathophysiological pathways contributing to the physiological response to LPS-induced injury. Pathophysiological pathways of LPS-induced lung injury are not only influenced by the type of the primary insult (e.g., pulmonary or extra-pulmonary) and presence of additional stimuli (e.g., mechanical ventilation), but also by time. As such, findings in animal models of LPS-induced lung injury may depend on the time point at which samples are obtained and physiological data are captured. This review summarizes the current evidence and highlights uncertainties on the molecular dynamics of LPS-induced lung injury in rodent models, encouraging researchers to take accurate timing of LPS-induced injury into account when designing experimental trials.

Long-Term Evaluation of Functional Outcomes Following Rat Volumetric Muscle Loss Injury and Repair.

Volumetric muscle loss (VML) injuries, by definition, exceed the endogenous repair capacity of skeletal muscle resulting in permanent structural and functional deficits. VML injuries present a significant burden for both civilian and military medicine. Despite progress, there is still considerable room for therapeutic improvement. In this regard, tissue-engineered constructs show promise for VML repair, as they provide an opportunity to introduce both scaffolding and cellular components. We have pioneered the development of a tissue-engineered muscle repair (TEMR) technology created by seeding muscle progenitor cells onto a porcine-derived bladder acellular matrix followed by cyclic stretch preconditioning before implantation. Our work to date has demonstrated significant functional repair (60-90% functional recovery) in progressively larger rodent models of VML injury following TEMR implantation. Notwithstanding this success, TEMR implantation in cylindrically shaped VML injuries in the tibialis anterior (TA) muscle was associated with more variable functional outcomes than has been observed in sheet-like muscles such as the latissimus dorsi. In fact, previous observations documented a dichotomy of responses following TEMR implantation in a rodent TA VML injury model; with an ≈61% functional improvement observed in fewer than half (46%) of TEMR-implanted animals at 12 weeks postinjury. This current study builds directly from those observations as we modified the geometry of both the VML injury and the TEMR construct to determine if improved matching of the implanted TEMR construct to the surgically created VML injury resulted in increased functional recovery posttreatment. Following these modifications, we observed a comparable degree of functional improvement in a larger proportion of animals (≈67%) that was durable up to 24 weeks post-TEMR implantation. Moreover, in ≈25% of all TEMR-implanted animals, functional recovery was virtually complete (TEMR max responders), and furthermore, the functional recovery in all 67% of responding animals was accompanied by the presence of native-like muscle properties within the repaired TA muscle, including fiber cross-sectional area, fiber type, vascularization, and innervation. This study emphasizes the importance of tuning the application of tissue engineering technology platforms to the specific requirements of diverse VML injuries to improve functional outcomes. Impact Statement This report confirms and extends previous observations with our implantable tissue-engineered technology platform for repair of volumetric muscle loss (VML) injuries. Based on our prior work, we addressed factors hypothesized to be responsible for significant outcome variability following treatment of VML injuries in a rat tibialis anterior model. Through customization of the muscle repair technology to a specific VML injury, we were able to significantly increase the frequency at which functional recovery occurred, and furthermore, demonstrate durability out to 6 months. In addition, the enhanced biomimetic qualities of repaired muscle tissue were associated with the most robust functional outcomes.

Neuroprotective effects of a ketogenic diet in combination with exogenous ketone salts following acute spinal cord injury.

We have previously shown that induction of ketosis by ketogenic diet (KD) conveyed neuroprotection following spinal cord injury in rodent models, however, clinical translation may be limited by the slow raise of ketone levels when applying KD in the acute post-injury period. Thus we investigated the use of exogenous ketone supplementation (ketone sodium, KS) combined with ketogenic diet as a means rapidly inducing a metabolic state of ketosis following spinal cord injury in adult rats. In uninjured rats, ketone levels increased more rapidly than those in rats with KD alone and peaked at higher levels than we previously demonstrated for the KD in models of spinal cord injury. However, ketone levels in KD + KS treated rats with SCI did not exceed the previously observed levels in rats treated with KD alone. We still demonstrated neuroprotective effects of KD + KS treatment that extend our previous neuroprotective observations with KD only. The results showed increased neuronal and axonal sparing in the dorsal corticospinal tract. Also, better performance of forelimb motor abilities were observed on the Montoya staircase (for testing food pellets reaching) at 4 and 6 weeks post-injury and rearing in a cylinder (for testing forelimb usage) at 6 and 8 weeks post-injury. Taken together, the findings of this study add to the growing body of work demonstrating the potential benefits of inducing ketosis following neurotrauma. Ketone salt combined with a ketogenic diet gavage in rats with acute spinal cord injury can rapidly increase ketone body levels in the blood and promote motor function recovery. This study was approved by the Animal Care Committee of the University of British Columbia (protocol No. A14-350) on August 31, 2015.

The Positive Allosteric Modulator of α2/3-Containing GABAA Receptors, KRM-II-81, Is Active in Pharmaco-Resistant Models of Epilepsy and Reduces Hyperexcitability after Traumatic Brain Injury.

The imidizodiazepine, 5-(8-ethynyl-6-(pyridin-2-yl)-4H-benzo[f]imidazo[1,5-a][1,4]diazepin-3-yl)oxazole (KRM-II-81), is selective for α2/3-containing GABAA receptors. KRM-II-81 dampens seizure activity in rodent models with enhanced efficacy and reduced motor-impairment compared with diazepam. In the present study, KRM-II-81 was studied in assays designed to detect antiepileptics with improved chances of impacting pharmaco-resistant epilepsies. The potential for reducing neural hyperactivity weeks after traumatic brain injury was also studied. KRM-II-81 suppressed convulsions in corneal-kindled mice. Mice with kainate-induced mesial temporal lobe seizures exhibited spontaneous recurrent hippocampal paroxysmal discharges that were significantly reduced by KRM-II-81 (15 mg/kg, orally). KRM-II-81 also decreased convulsions in rats undergoing amygdala kindling in the presence of lamotrigine (lamotrigine-insensitive model) (ED50 = 19 mg/kg, i.p.). KRM-II-81 reduced focal and generalized seizures in a kainate-induced chronic epilepsy model in rats (20 mg/kg, i.p., three times per day). In mice with damage to the left cerebral cortex by controlled-cortical impact, enduring neuronal hyperactivity was dampened by KRM-II-81 (10 mg/kg, i.p.) as observed through in vivo two-photon imaging of layer II/III pyramidal neurons in GCaMP6-expressing transgenic mice. No notable side effects emerged up to doses of 300 mg/kg KRM-II-81. Molecular modeling studies were conducted: docking in the binding site of the α1β3γ2L GABAA receptor showed that replacing the C8 chlorine atom of alprazolam with the acetylene of KRM-II-81 led to loss of the key interaction with α1His102, providing a structural rationale for its low affinity for α1-containing GABAA receptors compared with benzodiazepines such as alprazolam. Overall, these findings predict that KRM-II-81 has improved therapeutic potential for epilepsy and post-traumatic epilepsy. SIGNIFICANCE STATEMENT: We describe the effects of a relatively new orally bioavailable small molecule in rodent models of pharmaco-resistant epilepsy and traumatic brain injury. KRM-II-81 is more potent and generally more efficacious than standard-of-care antiepileptics. In silico docking experiments begin to describe the structural basis for the relative lack of motor impairment induced by KRM-II-81. KRM-II-81 has unique structural and anticonvulsant effects, predicting its potential as an improved antiepileptic drug and novel therapy for post-traumatic epilepsy.