Biology subjects

Thomas, T. C.

Publications and source records attributed to Thomas, T. C..

7 recordsLinked to original sources

bioRxiv2024

Cerebral Microvascular Density, Permeability of the Blood-Brain Barrier, and Neuroinflammatory Responses Indicate Early Aging Characteristics in a Marfan Syndrome Mouse Model

Marfan Syndrome (MFS) is a connective tissue disorder due to mutations in fibrillin-1 (Fbn1), where a Fbn1 missense mutation (Fbn1C1039G/+) can result in systemic increases in the bioavailability and signaling of transforming growth factor-{beta} (TGF-{beta}). In a well-established mouse model of MFS (Fbn1C1041G/+), pre-mature aging of the aortic wall and the progression of aortic root aneurysm are observed by 6-months-of-age. TGF-{beta} signaling has been implicated in cerebrovascular dysfunction, loss of blood-brain barrier (BBB) integrity, and age-related neuroinflammation. We have reported that pre-mature vascular aging in MFS mice could extend to cerebrovasculature, where peak blood flow velocity in the posterior cerebral artery (PCA) of 6-month-old (6M) MFS mice was reduced, similarly to 12-month-old (12M) control mice. Case studies of MFS patients have documented neurovascular manifestations, including intracranial aneurysms, stroke, arterial tortuosity, as well as headaches and migraines, with reported incidence of pain and chronic fatigue. Despite these significant clinical observations, investigation into cerebrovascular dysfunction and neuropathology in MFS remains limited. Using 6M-control (C57BL/6) and 6M-MFS (Fbn1C1041G/+) and healthy 12M-control male and female mice, we test the hypothesis that abnormal Fbn1 protein expression is associated with altered cerebral microvascular density, BBB permeability, and neuroinflammation in the PCA-perfused hippocampus, all indicative of a pre-mature aging brain phenotype. Using Glut1 staining, 6M-MFS mice and 12M-CTRL similarly present decreased microvascular density in the dentate gyrus (DG), cornu ammonis 1 (CA1), and cornu ammonis 3 (CA3) regions of the hippocampus. 6M-MFS mice exhibit increased BBB permeability in the DG, CA1, and CA3 as evident by Immunoglobulin G (IgG) staining, which was more comparable to 12M-CTRL mice. 6M-MFS mice show a higher number of microglia in the hippocampus compared to age-matched control mice, a pattern resembling that of 12M-CTRL mice. This study represents the first known investigation into neuropathology in a mouse model of MFS and indicates that the pathophysiology underlying MFS leads to a systemic pre-mature aging phenotype. This study is crucial for identifying and understanding MFS-associated neurovascular and neurological abnormalities, underscoring the need for research aimed at improving the quality of life and managing pre-mature aging symptoms in MFS and related connective tissue disorders.

neuroscience↗

bioRxiv2024

Freezing Diluted Bovine Serum Albumin Standards Does Not Significantly Affect Standard Curves

Total protein isolation followed by quantitation is a common protocol in many laboratories. Quantitation is often done using a colorimetric assay such as the bicinchoninic acid (BCA) assay in which a change in the color of the BCA reagent is related to protein concentration. Extracted protein samples are compared to a standard curve made with dilutions of a protein standard such as bovine serum albumin (BSA) to determine their concentrations. A series of experiments was designed to determine the most reproducible and accurate method for quantifying protein concentrations of samples in an experimental series over time. The effect of freezing on diluted standards was investigated. Standards were frozen at -20{degrees}C or -80{degrees}C and serially thawed and refrozen up to three times prior to their use in a BCA assay. Thawing and refreezing the standards had no significant effect on protein concentration and the resulting standard curves. Inter-person and intra-person variability in the preparation of standards was also investigated. Protein concentration differences due to inter-person and intra-person variability were greater than protein concentration variability resulting from freezing and thawing, regardless of the freezing temperature. The most reproducible and accurate method for determining the protein concentration of extracted samples in an experimental series over time is diluting a large batch of BSA standards and freezing them at either -20{degrees}C or -80{degrees}C. Reproducibility was maintained with up to three freeze-thaws. HighlightsO_LIFreezing diluted BSA standards at either -20{degrees}C or -80{degrees}C and thawing and refreezing them up to three times does not significantly alter their protein concentrations. C_LIO_LIInter-person variability in standard curves is greater than intra-person variability, and controlling for developer decreases variability in most cases. C_LIO_LIThere is more consistency in standard curves when a single batch of diluted standards is aliquoted and frozen at either -20{degrees}C or -80{degrees}C and thawed up to three times than when the same investigator or different investigators make fresh standards before each assay. C_LI

cell biology↗

bioRxiv2023

In Vivo Phenotypic Vascular Dysfunction Extends Beyond the Aorta in a Mouse Model for Fibrillin-1 (FBN1) Mutation

In individuals with Marfan Syndrome (MFS), fibrillin-1 gene (FBN1) mutations can lead to vascular wall weakening and dysfunction. The experimental mouse model of MFS (FBN1C1041G/+) has been advantageous in investigating MFS-associated life-threatening aortic aneurysms. Although the MFS mouse model presents an accelerated-aging phenotype in elastic organs (e.g., lung, skin), the impact of FBN1 mutations on other central and peripheral arteries function and structure with the consideration of the impact of sex remains underexplored. In this study, we investigate if FBN1 mutation contributes to sex-dependent alterations in central and cerebral vascular function similar to phenotypic changes associated with normal aging in healthy control mice. In vivo ultrasound imaging of central and cerebral vasculature was performed in 6-month-old male and female MFS and C57BL/6 mice and sex-matched 12-month-old (middle-aged) healthy control mice. Our findings confirm aortic enlargement (aneurysm) and wall stiffness in MFS mice, but with exacerbation in male diameters. Coronary artery blood flow velocity (BFV) in diastole was not different but left pulmonary artery BFV was decreased in MFS and 12-month-old control mice regardless of sex. At 6 months of age, MFS male mice show decreased posterior cerebral artery BFV as compared to age-matched control males, with no difference observed between female cohorts. Reduced mitral valve early-filling velocities were indicated in MFS mice regardless of sex. Male MFS mice also demonstrated left ventricular hypertrophy. Overall, these results underscore the significance of biological sex in vascular function and structure in MFS mice, while highlighting a trend of pre-mature vascular aging phenotype in MFS mice that is comparable to phenotypes observed in older healthy controls.

physiology↗

bioRxiv2023

Effects of isoflurane and urethane anesthetics on glutamate neurotransmission in rat brain using in vivo amperometry

Aspects of glutamate neurotransmission implicated in normal and pathological conditions are often evaluated using in vivo recording paradigms in rats anesthetized with isoflurane or urethane. Urethane and isoflurane anesthesia influence glutamate neurotransmission through different mechanisms; however real-time outcome measures of potassium chloride (KCl)-evoked glutamate overflow and glutamate clearance kinetics have not been compared within and between regions of the brain. In the following experiments, in vivo amperometric recordings of KCl-evoked glutamate overflow and glutamate clearance kinetics (uptake rate and T80) in the cortex, hippocampus and thalamus were performed using glutamate-selective microelectrode arrays (MEAs) in young adult male, Sprague-Dawley rats anesthetized with isoflurane or urethane. Potassium chloride (KCl)-evoked glutamate overflow was similar under urethane and isoflurane anesthesia in all brain regions studied. Analysis of glutamate clearance determined that the uptake rate was significantly faster (53.2%, p<0.05) within the thalamus under urethane compared to isoflurane, but no differences were measured in the cortex or hippocampus. Under urethane, glutamate clearance parameters were region dependent, with significantly faster glutamate clearance in the thalamus compared to the cortex but not the hippocampus (p<0.05). No region dependent differences were measured for glutamate overflow using isoflurane. These data support that amperometric recordings of glutamate under isoflurane and urethane anesthesia result in mostly similar and comparable data. However, certain parameters of glutamate uptake vary based on choice of anesthesia and brain region. Special considerations must be given to these areas when considering comparison to previous literature and when planning future experiments. HighlightsO_LIKCl-evoked glutamate overflow was similar under urethane and isoflurane anesthesia C_LIO_LIGlutamate clearance parameters were similar under both anesthesias in the cortex and hippocampus but not the thalamus C_LIO_LIGlutamate clearance kinetics differ between brain regions when anesthetized with urethane. C_LIO_LIExperimental design, brain region of interest, and outcome parameters of glutamate clearance should be considered when designing anesthetized amperometry recordings of glutamate. C_LI

neuroscience↗

bioRxiv2023

Aging with TBI vs. Aging: 6-month temporal profiles for neuropathology and astrocyte activation converge in behaviorally relevant thalamocortical circuitry of male and female rats

Traumatic brain injury (TBI) manifests late-onset and persisting clinical symptoms with implications for sex differences and increased risk for the development of age-related neurodegenerative diseases. Few studies have evaluated chronic temporal profiles of neuronal and glial pathology that include sex as a biological variable. After experimental diffuse TBI, late-onset and persisting somatosensory hypersensitivity to whisker stimulation develops at one-month post-injury and persists to at least two months post-injury in male rats, providing an in vivo model to evaluate the temporal profile of pathology responsible for morbidity. Whisker somatosensation is dependent on signaling through the thalamocortical relays of the whisker barrel circuit made up of glutamatergic projections between the ventral posteromedial nucleus of the thalamus (VPM) and primary somatosensory barrel cortex (S1BF) with inhibitory (GABA) innervation from the thalamic reticular nucleus (TRN) to the VPM. To evaluate the temporal profiles of pathology, male and female Sprague Dawley rats (n = 5-6/group) were subjected to sham surgery or midline fluid percussion injury (FPI). At 7-, 56-, and 168-days post-injury (DPI), brains were processed for amino-cupric silver stain and glial fibrillary acidic protein (GFAP) immunoreactivity, where pixel density of staining was quantified to determine the temporal profile of neuropathology and astrocyte activation in the VPM, S1BF, and TRN. FPI induced significant neuropathology in all brain regions at 7 DPI. At 168 DPI, neuropathology remained significantly elevated in the VPM and TRN, but returned to sham levels in the S1BF. GFAP immunoreactivity was increased as a function of FPI and DPI, with an FPI x DPI interaction in all regions and an FPI x Sex interaction in the S1BF. The interactions were driven by increased GFAP immunoreactivity in shams over time in the VPM and TRN. In the S1BF, GFAP immunoreactivity increased at 7 DPI and declined to age-matched sham levels by 168 DPI, while GFAP immunoreactivity in shams significantly increased between 7 and 168 days. The FPI x Sex interaction was driven by an overall greater level of GFAP immunoreactivity in FPI males compared to FPI females. Increased GFAP immunoreactivity was associated with an increased number of GFAP-positive soma, predominantly at 7 DPI. Overall, these findings indicate that FPI, time post-injury, sex, region, and aging with injury differentially contribute to chronic changes in neuronal pathology and astrocyte activation after diffuse brain injury. Thus, our results highlight distinct patterns of pathological alterations associated with the development and persistence of morbidity that supports chronic neuropathology, especially within the thalamus. Further, data indicate a convergence between TBI-induced and age-related pathology where further investigation may reveal a role for divergent astrocytic phenotypes associated with increased risk for neurodegenerative diseases.

neuroscience↗

bioRxiv2020

Spatial distribution of neuropathology and neuroinflammation elucidate the biomechanics of fluid percussion injury

Diffuse brain injury is better described as multi-focal, where pathology can be found adjacent to seemingly uninjured neural tissue. In experimental diffuse brain injury, pathology and pathophysiology have been reported far more lateral than predicted by the impact site. Finite element biomechanical models of diffuse brain injury predict regions of maximum stress and strain. However, the application of a skull with uniform thickness may mask the pathophysiology due to varying thickness of human and animal skulls. Force applied to the intact skull would diffuse the forces, whereas forces applied through an open skull are distributed along paths of least resistance within and then exiting the skull. We hypothesized that the local thickening of the rodent skull at the temporal ridges serves to focus the intracranial mechanical forces experienced during brain injury and generate predictable pathology in underlying cortical tissue. We demonstrated local thickening of the skull at the temporal ridges using contour analysis of coronal skull sections and oblique sectioning on MRI. After diffuse brain injury induced by midline fluid percussion injury (mFPI), pathological foci along the anterior-posterior length of cortex under the temporal ridges were evident acutely (1, 2, 7 days) and chronically (28 days) post-injury by deposition of argyophilic reaction product. Area CA3 of the hippocampus and lateral nuclei of the thalamus showed pathological change, suggesting that mechanical forces to or from the temporal ridges shear subcortical regions. A proposed model of mFPI biomechanics suggests that injury force vectors reflect off the skull base and radiate toward the temporal ridge due to the material properties of the skull based on thickness, thereby injuring ventral thalamus, dorsolateral hippocampus, and sensorimotor cortex. Surgically thinning the temporal ridge prior to injury reduced the injury-induced inflammation in sensorimotor cortex. These data build evidence for the temporal ridges of the rodent skull to contribute to the observed pathology, whether by focusing extracranial forces to enter the cranium or intracranial forces to escape the cranium. Pre-clinical investigations can take advantage of the predicted pathology to explore injury mechanisms and treatment efficacy. HighlightsO_LIThe temporal ridge is 75% thicker than the adjacent skull of the rodent C_LIO_LIExperimental diffuse TBI neuropathology occurs beneath the length of the temporal ridge C_LIO_LINeuropathology encompasses sensorimotor cortex, somatosensory thalamus, and dorsolateral hippocampus C_LIO_LIProposed mechanism of biomechanical injury forces include the temporal ridge C_LI

neuroscience↗

bioRxiv2019

Diffuse TBI-induced expression of anxiety-like behavior coincides with altered glutamatergic function, TrkB protein levels and region-dependent pathophysiology in amygdala circuitry

Up to 50% of traumatic brain injury (TBI) survivors demonstrate persisting affective symptoms indicative of limbic system dysregulation, yet the pathophysiology underlying the symptoms is unclear. We hypothesize that TBI-induced pathophysiologic changes within distinct amygdala nuclei contribute to the expression of late-onset anxiety-like behavior. Adult, male Sprague-Dawley rats underwent midline fluid percussion injury or sham surgery. Anxiety-like behavior was assessed at 7 and 28 days post-injury (DPI) followed by assessment of real-time glutamate neurotransmission in the basolateral amygdala (BLA) and central nucleus of the amygdala (CeA) using glutamate-selective microelectrode arrays. In separate animal cohorts, the presence of neuropathology, astrocytosis, and microglial activation were assessed at 1, 7, and 28DPI. Protein levels of glutamatergic transporters (Glt-1, GLAST) and presynaptic modulators of glutamate release (mGluR2, TrkB, BDNF, and glucocorticoid receptors) were quantified using automated capillary western techniques at 28DPI. The expression of anxiety-like behavior at 28DPI coincided with decreased glutamate release and slower glutamate clearance in the CeA, not BLA. Changes in glutamate neurotransmission were independent of protein levels of glutamate transporters and mGluR2 receptors, neuropathology, and astrocytosis. At 1DPI, microglia in the CeA demonstrated a neuroinflammatory response. BDNF and TrkB were decreased at 28DPI in the amygdala. These data indicate that diffuse axonal injury instigates sequences of molecular, structural and functional changes in the amygdala that contribute to circuit dysregulation relevant to the expression of affective disorders. Translationally, diffuse axonal injury can influence severity and incidence of affective symptoms and should be addressed in the history of patients with affective disorders.

neuroscience↗