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Marfan syndrome (MFS) is a connective tissue disorder (CTD) associated with mutations in fibrillin-1 (Fbn1), leading to systemic increases in transforming growth factor-β (TGF-β) availability and signaling that induces an accelerated vascular aging phenotype with known peripheral vascular dysfunction by 6-months (6M) of age in Fbn1 C1041G/+ (MFS) mice. TGF-β has been implicated in cerebrovascular dysfunction, loss of blood-brain barrier (BBB) integrity, age-related neuroinflammation and neurological deficits. Improvements in MFS treatment and life expectancy have uncovered a risk for cerebrovascular complications such as stroke, and cerebral aneurysm, predominantly in the posterior cerebral artery (PCA). CTDs have shown unmasking events via mild traumatic brain injury (mTBI), where patients display increased severity and recovery times. Cerebrovascular dysfunction and neuropathology have been minimally investigated in MFS. We hypothesized that abnormal Fbn1 protein expression is associated with a sex-dependent accelerated aging phenotype in the aorta, cerebrovasculature, BBB permeability, neuroinflammation, behavior, and glutamate homeostasis, leaving the brain more vulnerable to mTBI. Male and female 6M MFS, 6M C57BL/6 (WT) control, and middle-aged 12M WT mice were utilized. In vivo ultrasound imaging demonstrated increased aortic root diameters and stiffness in MFS mice compared to 6M WT mice, like 12M WT, where MFS males were affected more than females. Aortic results were associated with decreased PCA blood flow velocity in MFS males, where MFS females were similar to 6 and 12M WT female mice. In 6M MFS mice, Evans blue extravasation and Immunoglobulin G staining demonstrated increased BBB permeability in the hippocampus to large and small molecules, respectively, as compared to WT and like 12M WT. In adjacent sections, iba-1 stained cells had morphologies indicative of reactive microglia. Acute glial activation and BBB permeability can disrupt glutamate homeostasis, where in vivo electrochemical recordings in the hippocampus showed slower glutamate clearance and elevated baseline glutamate concentrations in MFS and 12M WT mice. Higher neurobehavioral severity scale (NSS) scores in 6M MFS mice suggested neurobehavioral alterations that were more like 12M WT. Midline fluid percussion was used to induce mTBI, where 6M MFS male mice and both female groups required a 15% lower pressure to induce mTBI righting reflex times (5-10 minutes) compared to 6M WT male mice. Mice were evaluated at 1-day post injury, where BBB permeability to large and small molecules and microglia activation were increased, glutamate clearance was slowed, and NSS scores were increased compared to 6M WT and were similar to 6M MFS and 12M WT mice. These novel findings signify MFS as a disease of accelerated cerebrovascular aging phenotype and demonstrate potential vulnerability to injury, where chronic TGF-β dysregulation may be a critical regulator of age-related pathology. Valley_Research_Partnership-P1A-5012, NIH-R15HL145646, NIH-R01NS100793, Midwestern Graduate Funds 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.

Transforming Growth Factor-β (TGF-β) is a multifunctional cytokine expressed in virtually every cell type of the human body. It has various roles throughout the body and is heavily reliant on cellular and environmental context, with major roles in extracellular matrix (ECM) turnover and inflammation. TGF-β is upregulated after neurological insults including trauma and ischemic injury with implications for increased risk for aging-related phenotypes, such as neurodegeneration and cognitive decline. Neurotransmission in the hippocampus (HC) is largely glutamatergic, and exogenous TGF-β has been shown to decrease the expression of glutamate transporters, implicating TGF-β signaling as a potential mechanism for mediating extracellular glutamate homeostasis post-injury, where glutamate clearance dynamics have never been investigated. This study investigates how local TGF-β application to discrete regions within the lateral HC can modulate glutamatergic neurotransmission using in vivo amperometric recordings with glutamate selective microelectrode arrays (MEA) in adult (6M) male C57BL/6 mice (N=5) anesthetized with urethane (1.5g/kg, i.p.). We hypothesized that local application of exogenous TGF-β would yield a decrease in glutamate clearance rates, coinciding with an increase in glutamate clearance times. A MEA/double-barrel micropipette containing 100μM glutamate and 25ng/mL TFG-β loaded in separate barrels for individual application was placed in the dorsal lateral HC (AP: -2.75, ML: +/-3, DV: -1.5), where 4 local applications of glutamate (pre-TGF-β) were applied followed by a physiologically relevant concentration of TGF-β (400nL), followed by 4 local dose matched applications of glutamate (post-TBG-β). The MEA was then lowered (DV) for subsequent recordings at -2.2, -3, and -4mm. Post-TGF-β glutamate recordings were compared to pre-TGF-β glutamate recordings for changes in glutamate clearance kinetics, which demonstrated slower glutamate clearance at all depths measured in the lateral HC. Unexpectedly, TGF-β application yielded immediate and reproducible evoked glutamate overflow that reached peak concentrations of 6.28±1.74mM and remained elevated for 8.53±1.24s before returning to baseline levels in comparison to physiological saline. Results represent the first evidence that TGF-β can directly and indirectly modulate glutamate neurotransmission in the extracellular space of the HC. These are novel mechanisms (in addition to ECM turnover and neuroinflammation) by which injury-induced TGF-β can mediate post-injury neuropathological sequelae and symptoms after an acquired brain injury. T. Curry and T. Currier Thomas are co-senior authors. Midwestern Graduate Funds, NIH-R01NS100793. 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.

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 anin vivomodel 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 × DPI interaction in all regions and an FPI × 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 × 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.

Small volume chamber tensometric myography is a commonly used technique to evaluate the vascular contractility of small and large blood vessels in laboratory animals and small arteries isolated from human tissue. The technique allows researchers to maintain isolated blood vessels in a tightly controlled and standardized (near-physiological) setting, with the option of adjusting to various environmental factors, while challenging the isolated vessels with different pharmacological agents that can induce vasoconstriction or vasodilation. The myograph chamber also provides a platform to measure vascular reactivity in response to various hormones, inhibitors, and agonists that may impact the function of smooth muscle and endothelial layers separately or simultaneously. The blood vessel wall is a complex structure consisting of three different layers: the intima (endothelial layer), media (smooth muscle and elastin fibers), and adventitia (collagen and other connective tissue). To gain a clear understanding of the functional properties of each layer, it is critical to have access to an experimental platform and system that would allow for a combinational approach to study all three layers simultaneously. Such an approach demands access to a semi-physiological condition that would mimic the in vivo environment in an ex vivo setting. Small volume chamber tensometric myography has provided an ideal environment to evaluate the impact of environmental cues, experimental variables, or pharmacological agonists and antagonists on vascular properties. For many years, scientists have used the tensometric myograph technique to measure endothelial function and smooth muscle contractility in response to different agents. In this report, a small volume chamber tensometric myograph system is used to measure endothelial function in the isolated mouse aorta. This report focuses on how small volume chamber tensometric myography can be used to evaluate the functional integrity of the endothelium in small segments of a large artery such as the thoracic aorta.