Rat Model Of Brain Injury Research Articles

Investigation of the neuroprotective effect of Granisetron through SV2A and 5-HT3 modulation in a radiation-induced brain injury rat model

Investigation of the neuroprotective effect of Granisetron through SV2A and 5-HT3 modulation in a radiation-induced brain injury rat model

Non-Invasive Vagus Nerve Stimulation Improves Brain Lesion Volume and Neurobehavioral Outcomes in a Rat Model of Traumatic Brain Injury.

Abstract Traumatic brain injury (TBI) continues to be a major cause of death and disability worldwide. This study assessed the effectiveness of non-invasive vagus nerve stimulation (nVNS) in reducing brain lesion volume and improving neurobehavioral performance in a rat model of TBI. Animals were randomized into three experimental groups: (1) TBI with sham stimulation treatment (Control), (2) TBI treated with five lower doses (2-min) nVNS, and (3) TBI treated with five higher doses (2 × 2-min) nVNS. We used the gammaCore nVNS device to deliver stimulations. Magnetic resonance imaging studies were performed 1 and 7 days post-injury to confirm lesion volume. We observed smaller brain lesion volume in the lower dose nVNS group compared with the control group on days 1 and 7. The lesion volume for the higher dose nVNS group was significantly smaller than either the lower dose nVNS or the control groups on days 1 and 7 post-injury. The apparent diffusion coefficient differences between the ipsilateral and contralateral hemispheres on day 1 were significantly smaller for the higher dose (2 × 2 min) nVNS group than for the control group. Voxel-based morphometry analysis revealed an increase in the ipsilateral cortical volume in the control group caused by tissue deformation and swelling. On day 1, these abnormal volume changes were 13% and 55% smaller in the lower dose and higher dose nVNS groups, respectively, compared with the control group. By day 7, nVNS dampened cortical volume loss by 35% and 89% in the lower dose and higher dose nVNS groups, respectively, compared with the control group. Rotarod, beam walking, and anxiety performances were significantly improved in the higher-dose nVNS group on day 1 compared with the control group. The anxiety indices were also improved on day 7 post-injury compared with the control and the lower-dose nVNS groups. In conclusion, the higher dose nVNS (five 2 × 2-min stimulations) reduced brain lesion volume to a level that further refined the role of nVNS therapy for the acute treatment of TBI. Should nVNS prove effective in additional pre-clinical TBI models and later in clinical settings, it would have an enormous impact on the clinical practice of TBI in both civilian and military settings, as it can easily be adopted into routine clinical practice.

The effect of thioredoxin-1 in a rat model of traumatic brain injury depending on diurnal variation.

Traumatic brain injury (TBI) is a public health concern with limited treatment options because it causes a cascade of side effects that are the leading cause of hospital death. Thioredoxin is an enzyme with neuroprotective properties such as antioxidant, antiapoptotic, immune response modulator, and neurogenic, among others; it has been considered a therapeutic target for treating many disorders. The controlled cortical impact (CCI) model was used to assess the effect of recombinant human thioredoxin 1 (rhTrx1) (1 μg/2 μL, intracortical) on rats subjected to TBI at two different times of the light-dark cycle (01:00 and 13:00h). We analyzed the food intake, body weight loss, motor coordination, pain perception, and histology in specific hippocampus (CA1, CA2, CA3, and Dental Gyrus) and striatum (caudate-putamen) areas. Body weight loss, reduced food intake, spontaneous pain, motor impairment, and neuronal damage in specific hippocampus and striatum regions are more evident in rats subjected to TBI in the light phase than in the dark phase of the cycle and in groups that did not receive rhTrx1 or minocycline (as positive control). Three days after TBI, there is a recovery in body weight, food intake, motor impairment, and pain, which is more pronounced in the rats subjected to TBI at the dark phase of the cycle and those that received rhTrx1 or minocycline. Knowing the time of day a TBI occurs in connection to the neuroprotective mechanisms of the immune response in diurnal variation and the usage of the Trx1 protein might have a beneficial therapeutic impact in promoting quick recovery after a TBI.

Cerebral Glutamate Alterations Using Chemical Exchange Saturation Transfer Imaging in a Rat Model of Lipopolysaccharide-Induced Sepsis.

Glutamate-weighted chemical exchange saturation transfer (GluCEST) is a useful imaging tool to detect glutamate signal alterations caused by neuroinflammation. This study aimed to visualize and quantitatively evaluate hippocampal glutamate alterations in a rat model of sepsis-induced brain injury using GluCEST and proton magnetic resonance spectroscopy (1H-MRS). Twenty-one Sprague Dawley rats were divided into three groups (sepsis-induced groups (SEP05, n = 7 and SEP10, n = 7) and controls (n = 7)). Sepsis was induced through a single intraperitoneal injection of lipopolysaccharide (LPS) at a dose of 5 mg/kg (SEP05) or 10 mg/kg (SEP10). GluCEST values and 1H-MRS concentrations in the hippocampal region were quantified using conventional magnetization transfer ratio asymmetry and a water scaling method, respectively. In addition, we examined immunohistochemical and immunofluorescence staining to observe the immune response and activity in the hippocampal region after LPS exposure. The GluCEST and 1H-MRS results showed that GluCEST values and glutamate concentrations were significantly higher in sepsis-induced rats than those in controls as the LPS dose increased. GluCEST imaging may be a helpful technique for defining biomarkers to estimate glutamate-related metabolism in sepsis-associated diseases.

Caloric adequacy normalizes brain tissue protein turnover in a rat brain injury model

Traumatic brain injury (TBI) is a leading cause for hospitalization in America, often having long lasting effects including increased morbidity, mortality and cognitive deficits post recovery. Based on Standard of Care (SOC) treatment, TBI patients are grossly underfed, yet it is unclear how underfeeding effects protein turnover post-TBI. Thus, we designed a study to elucidate the effects of TBI and caloric restriction (CR) on protein dynamics post-TBI. Hypothesis: We hypothesize that TBI would elicit an increased fractional synthesis rate (FSR) in all brain tissue as compared to control, ad libitum (AL) fed animals, while CR would suppress FSR. Thus, we predicted that the CR+TBI animals would have an elevated FSR over CR control but suppressed compared to AL+TBI animals. Methods: Deuterium oxide was used to measure FSR 13-day post TBI in multiple tissues [skeletal muscle, heart, right and left cerebellum, right and left cortex and hippocampus] of Sprague Dawley rats, AL fed control (AL-Con: n=3), AL+TBI (n=6), or CR+TBI (n=5) and CR-Control (n=6). Tissue homogenates were fractionated to isolate the mitochondrial fraction, and cytoplasmic/sarcoplasmic fraction. Pentafluorobenzyl bromide derivatized alanine samples were measured by GC-MS to evaluate ions with m/z 448, 449, 450. FSR was determined using the m/z ion ratios and MIDA. We found that TBI significantly increased FSR over control animals in the left and right cortex (p≤0.05) and suppressed FSR in the cerebellum (p≤0.05). We found that CR in conjunction with TBI caused an increased FSR in the mitochondrial, cytosolic, and whole tissue of the cortical regions of the brain compared to CR control animals (p≤0.05). The increased FSR found with the CR-TBI group was mitigated with ad lib feeding, exhibiting caloric adequacy mitigates the effects of TBI in cortical and cerebral brain regions. This research shows evidence that caloric restriction has a negative impact on brain healing post-TBI. This project was funded by a grant to GAB from the PAC 12 athletic association (CAL-Brooks-17-02) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Dilation of the superior sagittal sinus detected in rat model of mild traumatic brain injury using 1 T magnetic resonance imaging.

Mild traumatic brain injury (mTBI) is a common injury that can lead to temporary and, in some cases, life-long disability. Magnetic resonance imaging (MRI) is widely used to diagnose and study brain injuries and diseases, yet mTBI remains notoriously difficult to detect in structural MRI. mTBI is thought to be caused by microstructural or physiological changes in the function of the brain that cannot be adequately captured in structural imaging of the gray and white matter. However, structural MRIs may be useful in detecting significant changes in the cerebral vascular system (e.g., the blood-brain barrier (BBB), major blood vessels, and sinuses) and the ventricular system, and these changes may even be detectable in images taken by low magnetic field strength MRI scanners (<1.5T). In this study, we induced a model of mTBI in the anesthetized rat animal model using a commonly used linear acceleration drop-weight technique. Using a 1T MRI scanner, the brain of the rat was imaged, without and with contrast, before and after mTBI on post-injury days 1, 2, 7, and 14 (i.e., P1, P2, P7, and P14). Voxel-based analyses of MRIs showed time-dependent, statistically significant T2-weighted signal hypointensities in the superior sagittal sinus (SSS) and hyperintensities of the gadolinium-enhanced T1-weighted signal in the superior subarachnoid space (SA) and blood vessels near the dorsal third ventricle. These results showed a widening, or vasodilation, of the SSS on P1 and of the SA on P1-2 on the dorsal surface of the cortex near the site of the drop-weight impact. The results also showed vasodilation of vasculature near the dorsal third ventricle and basal forebrain on P1-7. Vasodilation of the SSS and SA near the site of impact could be explained by the direct mechanical injury resulting in local changes in tissue function, oxygenation, inflammation, and blood flow dynamics. Our results agreed with literature and show that the 1T MRI scanner performs at a level comparable to higher field strength scanners for this type of research.

Reactive oxygen species-responsive HET0016 prodrug-loaded liposomes attenuate neuroinflammation and improve neurological deficit in a rat model of juvenile traumatic brain injury.

The arachidonic acid pathway metabolite 20-hydroxyeicosatetraenoic acid (20-HETE) contributes to ischemia/reperfusion brain injury. Inhibition of 20-HETE formation can protect the developing brain from global ischemia. In previous studies, we have found that treatment with the 20-HETE synthesis inhibitor N-hydroxy-N-4-butyl-2-methylphenylformamidine (HET0016) can protect the immature brain from traumatic brain injury (TBI), but its hydrophobic nature limits its full potential. We designed a reactive oxygen species-responsive HET0016 prodrug, which consists of a thioketal link between HET0016 and stearyl alcohol (HET-TK-SA), and used the nanoprodrug strategy to successfully synthesize liposomes HET0016 prodrug liposomes (HPLs) to facilitate the application of HET0016 in protection from TBI. HPLs demonstrated spherical shape, size of about 127.8 nm, a zeta potential of -28.8 mv, a narrow particle size distribution and good stability. Male rats at postnatal day 16-17 underwent controlled cortical impact (CCI) followed by intravenous injection with vehicle or HET0016 (1 mg/kg, 2 h post-injury, once/day for 3 days). The results of the in vivo demonstrated that HPLs has good biosafety and can pass through the blood-brain barrier. Not only that compared with HET0016, HPLs better-inhibited inflammation and improved neuronal degeneration, which further led to lesion volume reduction, upgraded behavioral task performance, and ameliorated the degree of TBI impairment. Our results demonstrated HPLs could be a new strategy for juvenile TBI therapy.

Pharmacological Characterization of [18F]-FNM and Evaluation of NMDA Receptors Activation in a Rat Brain Injury Model

PurposeNMDA receptors (NMDARs) dysfunction plays a central role in the physiopathology of psychiatric and neurodegenerative disorders whose mechanisms are still poorly understood. The development of a PET (positron emission tomography) tracer able to selectively bind to the NMDARs intra-channel PCP site may make it possible to visualize NMDARs in an open and active state. We describe the in vitro pharmacological characterization of [18F]-fluoroethylnormemantine ([18F]-FNM) and evaluate its ability to localize activated NMDA receptors in a rat preclinical model of excitotoxicity.ProceduresThe affinity of the non-radioactive analog for the intra-channel PCP site was determined in a radioligand competition assay using [3H]TCP ([3H]N-(1-[thienyl]cyclohexyl)piperidine) on rat brain homogenates. Selectivity was also investigated by the displacement of specific radioligands targeting various cerebral receptors. In vivo brain lesions were performed using stereotaxic quinolinic acid (QA) injections in the left motor area (M1) of seven Sprague Dawley rats. Each rat was imaged with a microPET/CT camera, 40 min after receiving a dose of 30 MBq + / − 20 of [18F]-FNM, 24 and 72 h after injury. Nine non-injured rats were also imaged using the same protocol.ResultsFNM displayed IC50 value of 13.0 ± 8.9 µM in rat forebrain homogenates but also showed significant bindings on opioid receptors. In the frontal and left somatosensory areas, [18F]FNM PET detected a mean of 37% and 41% increase in [18F]FNM uptake (p < 0,0001) 24 and 72 h after QA stereotaxic injection, respectively, compared to the control group.ConclusionsIn spite of FNM’s poor affinity for NMDAR PCP site, this study supports the ability of this tracer to track massive activation of NMDARs in neurological diseases.

Administration of selective brain hypothermia using a simple cooling device in neonatal rats

BackgroundThe interruption of oxygen and blood supply to the newborn brain around the time of birth is a risk factor for hypoxic-ischemic encephalopathy and may lead to infant mortality or lifelong neurological impairments. Currently, therapeutic hypothermia, the cooling of the infant's head or entire body, is the only treatment to curb the extent of brain damage. New methodIn this study, we designed a focal brain cooling device that circulates cooled water at a steady state temperature of 19 ± 1 °C through a coil of tubing fitted onto the neonatal rat's head. We tested its ability to selectively decrease brain temperature and offer neuroprotection in a neonatal rat model of hypoxic-ischemic brain injury. ResultsOur method cooled the brain to 30–33 °C in conscious pups, while keeping the core body temperature approximately 3.2 °C warmer. Furthermore, the application of the cooling device to the neonatal rat model demonstrated a reduction in brain volume loss compared to pups maintained at normothermia and achieved a level of brain tissue protection the same as that of whole-body cooling. Comparison with existing methodsPrevailing methods of selective brain hypothermia are designed for adult animal models rather than for immature animals such as the rat as a conventional model of developmental brain pathology. Contrary to existing methods, our method of cooling does not require surgical manipulation or anaesthesia. ConclusionOur simple, economical, and effective method of selective brain cooling is a useful tool for rodent studies in neonatal brain injury and adaptive therapeutic interventions.

ATPIF1 alleviates oxygen glucose deprivation/reoxygenation-induced astrocyte injury in vitro: A rat model of ischemic brain injury.

The role of ATPIF1 in ischemic brain injury is rarely reported. This study explored the effect of ATPIF1 on astrocyte activity under oxygen glucose deprivation/reoxygenation (OGD/R). The study sample was randomly allocated into: 1) control group (blank control); 2) OGD/R group (hypoxia for 6 h/reoxygenation for 1 h); 3) siRNA negative control (NC) group (OGD/R model+siRNA NC); and 4) siRNA-ATPIF1 group (OGD/R model+siRNA-ATPIF1). The OGD/R cell model was established from Sprague Dawley (SD) rats to simulate ischemia/reperfusion injury. Cells in the siRNA-ATPIF1 group were treated with siATPIF1. Ultrastructural changes in the mitochondria were observed using transmission electron microscopy (TEM). Apoptosis, cell cycle, reactive oxygen species (ROS), and mitochondrial membrane potential (MMP) were detected with flow cytometry. The protein expression levels of nuclear factor kappa B (NF-κB), B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X (Bax), and caspase-3 were detected with western blot. In the model group, the cell structure and the ridge structure were destroyed, and mitochondria edema, outer membrane damage and vacuole-like lesions were observed. Compared with the control group, the OGD/R group had considerably increased apoptosis, G0/G1 phase, ROS content, MMP, and Bax, caspase-3 and NF-κB protein expression, as well as markedly decreased S phase and Bcl-2 protein expression. Compared with the OGD/R group, the siRNA-ATPIF1 group had considerably decreased apoptosis, G0/G1 phase, ROS content, MMP, and Bax, caspase-3 and NF-κB protein expression, as well as remarkably increased S phase and Bcl-2 protein expression. The inhibition of ATPIF1 may alleviate OGD/R-induced astrocyte injury by regulating the NF-κB signaling pathway, inhibiting apoptosis, and reducing the ROS content and MMP in the rat brain ischemic model.

Protective Effects of Melanocortins on Short-Term Changes in a Rat Model of Traumatic Brain Injury: Retraction.

Protective Effects of Melanocortins on Short-Term Changes in a Rat Model of Traumatic Brain Injury: Retraction.

Protective effect of breviscapine against brain injury induced by intrauterine inflammation in preterm rats and its mechanism

To study the protective effect of breviscapine against brain injury induced by intrauterine inflammation in preterm rats and its mechanism. A preterm rat model of brain injury caused by intrauterine inflammation was prepared by intraperitoneal injections of lipopolysaccharide in pregnant rats. The pregnant rats and preterm rats were respectively randomly divided into 5 groups: control, model, low-dose breviscapine (45 mg/kg), high-dose breviscapine (90 mg/kg), and high-dose breviscapine (90 mg/kg)+ML385 [a nuclear factor erythroid 2-related factor 2 (Nrf2) inhibitor, 30 mg/kg] (n=10 each). The number and body weight of the live offspring rats were measured for each group. Hematoxylin-eosin staining was used to observe the pathological morphology of the uterus and placenta of pregnant rats and the pathological morphology of the brain tissue of offspring rats. Immunofluorescent staining was used to measure the co-expression of ionized calcium binding adaptor molecule-1 (IBA-1) and nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) in the cerebral cortex of offspring rats. ELISA was used to measure the levels of interleukin-6 (IL-6), interleukin-8 (IL-8), and interleukin-1β (IL-1β) in the brain tissue of offspring rats. Western blotting was used to measure the expression of Nrf2 pathway-related proteins in the brain tissue of offspring rats. Pathological injury was found in the uterus, and placenta tissue of the pregnant rats and the brain tissue of the offspring rats, and severe microglia pyroptosis occurred in the cerebral cortex of the offspring rats in the model group. Compared with the control group, the model group had significant reductions in the number and body weight of the live offspring rats and the protein expression levels of Nrf2 and heme oxygenase-1 (HO-1) in the brain tissue of the offspring rats (P<0.05), but significant increases in the relative fluorescence intensity of the co-expression of IBA-1 and NLRP3, the levels of the inflammatory factors IL-6, IL-8, and IL-1β, and the protein expression levels of NLRP3 and caspase-1 in the brain tissue of the offspring rats (P<0.05). Compared with the model group, the breviscapine administration groups showed alleviated pathological injury of the uterus and placenta tissue of the pregnant rats and the brain tissue of the offspring rats, significant increases in the number and body weight of the live offspring rats and the protein expression levels of Nrf2 and HO-1 in the brain tissue of the offspring rats (P<0.05), and significant reductions in the relative fluorescence intensity of the co-expression of IBA-1 and NLRP3, the levels of the inflammatory factors IL-6, IL-8, and IL-1β, and the protein expression levels of NLRP3 and caspase-1 in the brain tissue of the offspring rats (P<0.05). The high-dose breviscapine group had a significantly better effect than the low-dose breviscapine (P<0.05). ML385 significantly inhibited the intervention effect of high-dose breviscapine (P<0.05). Breviscapine can inhibit inflammatory response in brain tissue of preterm rats caused by intrauterine inflammation by activating the Nrf2 pathway, and it can also inhibit microglial pyroptosis and alleviate brain injury.

Netrin-1 attenuates cerebral ischemia/reperfusion injury by limiting mitochondrial ROS and Ca2+ levels via activation of AKT phosphorylation and mitochondrial m-AAA protease AFG3L2.

Cerebral ischemia-reperfusion (I/R) injury as the consequence of revascularization after ischemic stroke is associated with mitochondrial dysfunction, oxidative stress, and neuron loss. In this study, we used a deprivation/reoxygenation (OGD/R) model to determine whether interactions between Netrin-1, AKT, and the mitochondrial AAA protease AFG3L2 could influence mitochondrial function in neurons after I/R. We found that Netrin-1 protects primary cortical neurons from OGD/R-induced cell death and regulates mitochondrial reactive oxygen species (ROS) and Ca2+ levels. The accumulation of mitochondrial calcium uniporter (MCU) subunits was monitored in cells by immunoblot analysis. Although the regulatory subunits MICU1 and MICU2 were relatively unaffected, the accumulation of the essential MCU regulator (EMRE) subunit was impaired. In OGD/R-induced cells, the 7kDa form of EMRE was significantly reduced. Netrin-1 inhibited the accumulation of EMRE and mitochondrial Ca2+ levels by upregulating AFG3L2 and AKT activation. Loss of AFG3L2 or inhibition of AKT increased levels of 7kDa EMRE. Moreover, overexpression of AKT increased the expression of AFG3L2 in Netrin-1-knockdown neurons after OGD/R. Our results demonstrate that Netrin-1 enhanced AFG3L2 protein expression via activation of AKT. We also observed that overexpression of Netrin-1 significantly reduced infarction size in an I/R-induced brain injury model in rats but not when AKT was inhibited. Our data suggest that AFG3L2 is a protein substrate of AKT and indicate that Netrin-1 attenuates cerebral I/R injury by limiting mitochondrial ROS and Ca2+ levels through activating AKT phosphorylation and AFG3L2.

Diffusion MRI approaches for investigating microstructural complexity in a rat model of traumatic brain injury

Our study explores the potential of conventional and advanced diffusion MRI techniques including diffusion tensor imaging (DTI), and single-shell 3-tissue constrained spherical deconvolution (SS3T-CSD) to investigate complex microstructural changes following severe traumatic brain injury in rats at a chronic phase. Rat brains after sham-operation or lateral fluid percussion (LFP) injury were scanned ex vivo in a 9.4 T scanner. Our region-of-interest-based approach of tensor-, and SS3T-CSD derived fixel-, 3-tissue signal fraction maps were sensitive to changes in both white matter (WM) and grey matter (GM) areas. Tensor-based measures, such as fractional anisotropy (FA) and radial diffusivity (RD), detected more changes in WM and GM areas as compared to fixel-based measures including apparent fiber density (AFD), peak FOD amplitude and primary fiber bundle density, while 3-tissue signal fraction maps revealed distinct changes in WM, GM, and phosphate-buffered saline (PBS) fractions highlighting the complex tissue microstructural alterations post-trauma. Track-weighted imaging demonstrated changes in track morphology including reduced curvature and average pathlength distal from the primary lesion in severe TBI rats. In histological analysis, changes in the diffusion MRI measures could be associated to decreased myelin density, loss of myelinated axons, and increased cellularity, revealing progressive microstructural alterations in these brain areas five months after injury. Overall, this study highlights the use of combined conventional and advanced diffusion MRI measures to obtain more precise insights into the complex tissue microstructural alterations in chronic phase of severe brain injury.

Abstract WMP118: Sex-specific And Dose-dependent Effects Of Drag-reducing Polymers On Post-traumatic Ischemia In Rats After Traumatic Brain Injury Of Different Severity

Introduction: Previously, we showed that drag-reducing polymers (DRP) improve hemodynamics in rat models of brain injury (TBI). Here we examined the dose- and sex-dependent DRP effects on post-TBI ischemia. Methods: In-vivo 2-photon microscopy over the rat parietal cortex was used to monitor the effect of DRP on microvascular perfusion and tissue oxygenation (NADH) and blood-brain barrier permeability. Lateral fluid-percussion TBI (1.5 ATA-moderate or 2.5 ATA-severe, 100 ms) was induced after baseline imaging and followed by 4 hours of monitoring. DRP injected at 1, 2, or 4 ppm. Data analysis was done by GraphPad Prism 7. Results: Moderate TBI progressively decreased microvascular circulation and hypoxia in the pericontusion zone (p&lt;0.05). The i.v. injection of DRP increased near-wall flow velocity and flow rate in arterioles, leading to an increase in the number of erythrocytes entering capillaries, enhancing capillary perfusion in a dose-dependent manner without distinguishable difference between males and females (p&lt;0.01). The severe TBI resulted in intracranial pressure increase (31±3.2 mmHg, p&lt;0.05), leading to microcirculation redistribution to non-nutritive microvascular shunt (MVS) flow and stagnation of capillary flow. Tissue hypoxia was more prominent, especially in male rats; the pericontusion zone was 25±5.2% larger than after moderate TBI (p&lt;0.01). Both were reverted by DRP in a dose-related manner with better efficiency in females (p&lt;0.01). After severe TBI, BBB degradation was faster and more prominent. DRP was more efficient in attenuating the progression of permeability increase in moderate TBI (p&lt;0.05). Conclusion: DRP at 4 ppm was most efficient, with a better effect on female rats. Supported by NIH/NINDS R01NS112808

Novel image analysis tool for rapid screening of cell morphology in preclinical animal models of disease

The field of cell biology has seen major advances in both cellular imaging modalities and the development of automated image analysis platforms that increase rigor, reproducibility, and throughput for large imaging data sets. However, there remains a need for tools that provide accurate morphometric analysis of single cells with complex, dynamic cytoarchitecture in a high-throughput and unbiased manner. We developed a fully automated image-analysis algorithm to rapidly detect and quantify changes in cellular morphology using microglia cells, an innate immune cell within the central nervous system, as representative of cells that exhibit dynamic and complex cytoarchitectural changes. We used two preclinical animal models that exhibit robust changes in microglia morphology: (1) a rat model of acute organophosphate intoxication, which was used to generate fluorescently labeled images for algorithm development; and (2) a rat model of traumatic brain injury, which was used to validate the algorithm using cells labeled using chromogenic detection methods. All ex vivo brain sections were immunolabeled for IBA-1 using fluorescence or diaminobenzidine (DAB) labeling, images were acquired using a high content imaging system and analyzed using a custom-built algorithm. The exploratory data set revealed eight statistically significant and quantitative morphometric parameters that distinguished between phenotypically distinct groups of microglia. Manual validation of single-cell morphology was strongly correlated with the automated analysis and was further supported by a comparison with traditional stereology methods. Existing image analysis pipelines rely on high-resolution images of individual cells, which limits sample size and is subject to selection bias. However, our fully automated method integrates quantification of morphology and fluorescent/chromogenic signals in images from multiple brain regions acquired using high-content imaging. In summary, our free, customizable image analysis tool provides a high-throughput, unbiased method for accurately detecting and quantifying morphological changes in cells with complex morphologies.

A simple method to isolate structurally and chemically intact brain vascular basement membrane for neural regeneration following traumatic brain injury

BackgroundThe brain vascular basement membrane (brain-VBM) is an important component of the brain extracellular matrix, and the three-dimensional structure of the cerebrovascular network nested with many cell-adhesive proteins may provide guidance for brain tissue regeneration. However, the potential of ability of brain-VBM to promote neural tissue regeneration has not been examined due to the technical difficulty of isolating intact brain-VBM.MethodsThe present study developed a simple, effective method to isolate structurally and compositionally intact brain-VBM. Structural and component properties of the brain-VBM were characterized to confirm the technique. Seed cells were cocultured with brain-VBM in vitro to analyze biocompatibility and neurite extension. An experimental rat model of focal traumatic brain injury (TBI) induced by controlled cortical impact were conducted to further test the tissue regeneration ability of brain-VBM.ResultsBrain-VBM isolated using genipin showed significantly improved mechanical properties, was easy to handle, supported high cell viability, exhibited strong cell adhesive properties, and promoted neurite extension and outgrowth. Further testing of the isolated brain-VBM transplanted at lesion sites in an experimental rat model of focal TBI demonstrated considerable promise for reconstructing a complete blood vessel network that filled in the lesion cavity and promoting repopulation of neural progenitor cells and neurons.ConclusionThe technique allows isolation of intact brain-VBM as a 3D microvascular scaffold to support brain tissue regeneration following TBI and shows considerable promise for the production of naturally-derived biomaterials for neural tissue engineering.Graphical

An end-end deep learning framework for lesion segmentation on multi-contrast MR images—an exploratory study in a rat model of traumatic brain injury

Traumatic brain injury (TBI) engenders traumatic necrosis and penumbra-areas of secondary neural injury which are crucial targets for therapeutic interventions. Segmenting manually areas of ongoing changes like necrosis, edema, hematoma, and inflammation is tedious, error-prone, and biased. Using the multi-parametric MR data from a rodent model study, we demonstrate the effectiveness of an end-end deep learning global-attention-based UNet (GA-UNet) framework for automatic segmentation and quantification of TBI lesions. Longitudinal MR scans (2h, 1, 3, 7, 14, 30, and 60days) were performed on eight Sprague-Dawley rats after controlled cortical injury was performed. TBI lesion and sub-regions segmentation was performed using 3D-UNet and GA-UNet. Dice statistics (DSI) and Hausdorff distance were calculated to assess the performance. MR scan variations-based (bias, noise, blur, ghosting) data augmentation was performed to develop a robust model.Training/validation median DSI for U-Net was 0.9368 with T2w and MPRAGE inputs, whereas GA-UNet had 0.9537 for the same. Testing accuracies were higher for GA-UNet than U-Net with a DSI of 0.8232 for the T2w-MPRAGE inputs.Longitudinally, necrosis remained constant while oligemia and penumbra decreased, and edema appearing around day 3 which increased with time. GA-UNet shows promise for multi-contrast MR image-based segmentation/quantification of TBI in large cohort studies.

Association of sub-acute changes in plasma amino acid levels with long-term brain pathologies in a rat model of moderate-severe traumatic brain injury.

Traumatic brain injury (TBI) induces a cascade of cellular alterations that are responsible for evolving secondary brain injuries. Changes in brain structure and function after TBI may occur in concert with dysbiosis and altered amino acid fermentation in the gut. Therefore, we hypothesized that subacute plasma amino acid levels could predict long-term microstructural outcomes as quantified using neurite orientation dispersion and density imaging (NODDI). Fourteen 8-10-week-old male rats were randomly assigned either to sham (n = 6) or a single moderate-severe TBI (n = 8) procedure targeting the primary somatosensory cortex. Venous blood samples were collected at days one, three, seven, and 60 post-procedure and NODDI imaging were carried out at day 60. Principal Component Regression analysis was used to identify time dependent plasma amino acid concentrations after in the subacute phase post-injury that predicted NODDI metric outcomes at day 60. The TBI group had significantly increased plasma levels of glutamine, arginine, alanine, proline, tyrosine, valine, isoleucine, leucine, and phenylalanine at days three-seven post-injury. Higher levels of several neuroprotective amino acids, especially the branched-chain amino acids (valine, isoleucine, leucine) and phenylalanine, as well as serine, arginine, and asparagine at days three-seven post-injury were also associated with lower isotropic diffusion volume fraction measures in the ventricles and thus lesser ventricular dilation at day 60. In the first such study, we examined the relationship between the long-term post-TBI microstructural outcomes across whole brain and the subacute changes in plasma amino acid concentrations. At days three to seven post-injury, we observed that increased plasma levels of several amino acids, particularly the branched-chain amino acids and phenylalanine, were associated with lesser degrees of ventriculomegaly and hydrocephalus TBI neuropathology at day 60 post-injury. The results imply that altered amino acid fermentation in the gut may mediate neuroprotection in the aftermath of TBI.

Reperfusion after hypoxia-ischemia exacerbates brain injury with compensatory activation of the anti- ferroptosis system: based on a novel rat model.

Hypoxic-ischemic encephalopathy, which predisposes to neonatal death and neurological sequelae, has a high morbidity, but there is still a lack of effective prevention and treatment in clinical practice. To better understand the pathophysiological mechanism underlying hypoxic-ischemic encephalopathy, in this study we compared hypoxic-ischemic reperfusion brain injury and simple hypoxic-ischemic brain injury in neonatal rats. First, based on the conventional Rice-Vannucci model of hypoxic-ischemic encephalopathy, we established a rat model of hypoxic-ischemic reperfusion brain injury by creating a common carotid artery muscle bridge. Then we performed tandem mass tag-based proteomic analysis to identify differentially expressed proteins between the hypoxic-ischemic reperfusion brain injury model and the conventional Rice-Vannucci model and found that the majority were mitochondrial proteins. We also performed transmission electron microscopy and found typical characteristics of ferroptosis, including mitochondrial shrinkage, ruptured mitochondrial membranes, and reduced or absent mitochondrial cristae. Further, both rat models showed high levels of glial fibrillary acidic protein and low levels of myelin basic protein, which are biological indicators of hypoxic-ischemic brain injury and indicate similar degrees of damage. Finally, we found that ferroptosis-related Ferritin (Fth1) and glutathione peroxidase 4 were expressed at higher levels in the brain tissue of rats with hypoxic-ischemic reperfusion brain injury than in rats with simple hypoxic-ischemic brain injury. Based on these results, it appears that the rat model of hypoxic-ischemic reperfusion brain injury is more closely related to the pathophysiology of clinical reperfusion. Reperfusion not only aggravates hypoxic-ischemic brain injury but also activates the anti-ferroptosis system.