Regional differences in Alzheimer's disease pathology confound behavioural rescue after amyloid-ß attenuation.

Failure of Alzheimer's disease clinical trials to improve or stabilize cognition has led to the need for a better understanding of the driving forces behind cognitive decline in the presence of active disease processes. To dissect contributions of individual pathologies to cognitive function, we used the TgF344-AD rat model, which recapitulates the salient hallmarks of Alzheimer's disease pathology observed in patient populations (amyloid, tau inclusions, frank neuronal loss, and cognitive deficits). scyllo-Inositol treatment attenuated amyloid-ß peptide in disease-bearing TgF344-AD rats, which rescued pattern separation in the novel object recognition task and executive function in the reversal learning phase of the Barnes maze. Interestingly, neither activities of daily living in the burrowing task nor spatial memory in the Barnes maze were rescued by attenuating amyloid-ß peptide. To understand the pathological correlates leading to behavioural rescue, we examined the neuropathology and in vivo electrophysiological signature of the hippocampus. Amyloid-ß peptide attenuation reduced hippocampal tau pathology and rescued adult hippocampal neurogenesis and neuronal function, via improvements in cross-frequency coupling between theta and gamma bands. To investigate mechanisms underlying the persistence of spatial memory deficits, we next examined neuropathology in the entorhinal cortex, a region whose input to the hippocampus is required for spatial memory. Reduction of amyloid-ß peptide in the entorhinal cortex had no effect on entorhinal tau pathology or entorhinal-hippocampal neuronal network dysfunction, as measured by an impairment in hippocampal response to entorhinal stimulation. Thus, rescue or not of cognitive function is dependent on regional differences of amyloid-ß, tau and neuronal network dysfunction, demonstrating the importance of staging disease in patients prior to enrolment in clinical trials. These results further emphasize the need for combination therapeutic approaches across disease progression.

Acute and chronic stage adaptations of vascular architecture and cerebral blood flow in a mouse model of TBI.

The 3D organization of cerebral blood vessels determines the overall capacity of the cerebral circulation to meet the metabolic requirements of the brain. Imaging methodologies which combine 3D microvascular structural imaging with blood flow quantification can shed light on the relationship between vascular structure and function, in health and disease. This study applies Arterial Spin Labeling (ASL) MRI with a hypercapnic challenge and ex vivo Serial Two-Photon Tomography (STPT) to examine the relationship between blood flow and vascular architecture following traumatic brain injury (TBI) in a mouse. Mice were exposed to a controlled cortical impact TBI and allowed to recover for either 1 day or 4 weeks. At each time point, ASL MRI was performed to quantify cerebral perfusion and the brain vasculature was imaged in 3D with STPT. Registration of ASL to STPT enabled flow changes to be related to the underlying microvascular structure in each ASL voxel. Hypoperfusion under rest and hypercapnia was observed both 1 day and 4 weeks post-TBI. Vessel density and vascular volume were reduced 1 day post-TBI, recovering by 4 weeks; however, the reorganized vasculature at the latter time point possessed an abnormal radial pattern. Our findings demonstrate functionally significant long-term changes in the vascular architecture following injury and illustrate why metrics beyond traditional measures of vessel density are required to understand the impact of vascular structure on function.

In vivo neurovascular response to focused photoactivation of Channelrhodopsin-2.

The rapid growth in the use of optogenetics for neuroscience applications is largely driven by two important advantages: highly specific cellular targeting through genetic manipulations; and precise temporal control of neuronal activation via temporal modulation of the optical stimulation. The difference between the most commonly used stimulation modalities, namely diffused (i.e. synchronous) and focused (i.e. asynchronous) stimulation has not been described. Furthermore, full realization of optogenetics' potential is hindered by our incomplete understanding of the cellular and network level response to photoactivation. Here we address these gaps by examining the neuronal and cerebrovascular responses to focused and diffuse photostimulation of channelrhodopsin in the Thy1-ChR2 mouse. We presented the responses of photoactivation via 470-nm fiber optic illumination (diffuse) alongside 458-nm raster-scan (focused) stimulation of the barrel field. Local field potentials (LFP) assessment of intracerebral electrophysiology and two-photon fluorescence microscopy measurements of red blood cell (RBC) speed (vRBC) in cortical penetrating vessels revealed ∼40% larger LFP responses (p = 0.05) and twice as large cerebrovascular responses (p = 0.002) under focused vs. diffuse photostimulation (focused: 1.64 ±â€¯0.84 mV LFP amplitude and 75 ±â€¯48% increase in vRBC; diffuse: 1.14 ±â€¯0.75 mV LFP amplitude and 35 ±â€¯23% increase in vRBC). Compared to diffuse photostimulation, focused photostimulation resulted in a ∼65% increase in the yield of cerebrovascular responses (73 ±â€¯10% for focused and 42 ±â€¯29% for diffuse photostimulation) and a doubling of the signal-to-noise ratio of the cerebrovascular response (20.9 ±â€¯14.7 for focused and 10.4 ±â€¯1.4 for diffuse photostimulation). These data reveal important advantages of focused optogenetic photoactivation, which can be easily integrated into single- or two-photon fluorescence microscopy platforms, as a means of assessing neuronal excitability and cerebrovascular reactivity, thus paving the way for broader application of optogenetics in preclinical models of CNS diseases.

Imaging the Effects of ß-Hydroxybutyrate on Peri-Infarct Neurovascular Function and Metabolism.

Background and Purpose- Recent evidence suggests great potential of metabolically targeted interventions for treating neurological disorders. We investigated the use of the endogenous ketone body ß-hydroxybutyrate (BHB) as an alternate metabolic substrate for the brain in the acute phase of ischemia because postischemic hyperglycemia and brain glucose metabolism elevation compromise functional recovery. Methods- We delivered BHB (or vehicle) 1 hour after ischemic insult induced by cortical microinjection of endothelin-1 in sensorimotor cortex of rats. Two days after ischemic insult, the rats underwent multimodal characterization of the BHB effects. We examined glucose uptake on 2-Deoxy-d-glucose chemical exchange saturation transfer magnetic resonance imaging, cerebral hemodynamics on continuous arterial spin labeling magnetic resonance imaging, resting-state field potentials by intracerebral multielectrode arrays, Neurological Deficit Score, reactive oxygen species production, and astrogliosis and neuronal death. Results- When compared with vehicle-administered animals, BHB-treated cohort showed decreased peri-infarct neuronal glucose uptake which was associated with reduced oxidative stress, diminished astrogliosis and neuronal death. Functional examination revealed ameliorated neuronal functioning, normalized perilesional resting perfusion, and ameliorated cerebrovascular reactivity to hypercapnia, suggesting improved functioning. Cellular and functional recovery of the neurogliovascular unit in the BHB-treated animals was associated with improved performance on the withdrawal test. Conclusions- We characterize the effects of the ketone body BHB administration at cellular and system levels after focal cortical stroke. The results demonstrate that BHB curbs the peri-infarct glucose-metabolism driven production of reactive oxygen species and astrogliosis, culminating in improved neurogliovascular and functional recovery.

Early-stage attenuation of phase-amplitude coupling in the hippocampus and medial prefrontal cortex in a transgenic rat model of Alzheimer's disease.

Alzheimer's disease (AD) is pathologically characterized by amyloid-ß peptide (Aß) accumulation, neurofibrillary tangle formation, and neurodegeneration. Preclinical studies on neuronal impairments associated with progressive amyloidosis have demonstrated some Aß-dependent neuronal dysfunction including modulation of gamma-aminobutyric acid-ergic signaling. The present work focuses on the early stage of disease progression and uses TgF344-AD rats that recapitulate a broad repertoire of AD-like pathologies to investigate the neuronal network functioning using simultaneous intracranial recordings from the hippocampus (HPC) and the medial prefrontal cortex (mPFC), followed by pathological analyses of gamma-aminobutyric acid (GABAA ) receptor subunits α1, α5, and δ, and glutamic acid decarboxylases (GAD65 and GAD67). Concomitant to amyloid deposition and tau hyperphosphorylation, low-gamma band power was strongly attenuated in the HPC and mPFC of TgF344-AD rats in comparison to those in non-transgenic littermates. In addition, the phase-amplitude coupling of the neuronal networks in both areas was impaired, evidenced by decreased modulation of theta band phase on gamma band amplitude in TgF344-AD animals. Finally, the gamma coherence between HPC and mPFC was attenuated as well. These results demonstrate significant neuronal network dysfunction at an early stage of AD-like pathology. This network dysfunction precedes the onset of cognitive deficits and is likely driven by Aß and tau pathologies. This article is part of the Special Issue "Vascular Dementia".

Venular degeneration leads to vascular dysfunction in a transgenic model of Alzheimer's disease.

Most patients with Alzheimer's disease exhibit accumulation of amyloid-ß peptide on leptomeningeal and cortical arterioles, or cerebral amyloid angiopathy, which is associated with impaired vascular reactivity and accelerated cognitive decline. Despite widespread recognition of the significance of vascular dysfunction in Alzheimer's disease aetiology and progression, much uncertainty still surrounds the mechanism underlying Alzheimer's disease vascular injury. Studies to date have focused on amyloid-ß-induced damage to capillaries and plaque-associated arterioles, without examining effects across the entire vascular bed. In the present study, we investigated the structural and functional impairment of the feeding arteriolar versus draining venular vessels in a transgenic murine Alzheimer's disease model, with a particular focus on the mural cell populations that dictate these vessels' contractility. Although amyloid-ß deposition was restricted to arterioles, we found that vascular impairment extended to the venules, which showed significant depletion of their mural cell coverage by the mid-stage of Alzheimer's disease pathophysiology. These structural abnormalities were accompanied by an abolishment of the normal vascular network flow response to hypercapnia: this functional impairment was so severe as to result in hypercapnia-induced flow decreases in the arterioles. Further pharmacological depletion of mural cells using SU6668, a platelet-derived growth factor receptor-ß antagonist, resulted in profound structural abnormalities of the cortical microvasculature, including vessel coiling and short-range looping, increased tortuosity of the venules but not of the arterioles, increased amyloid-ß deposition on the arterioles, and further alterations of the microvascular network cerebral blood flow response to hypercapnia. Together, this work shows hitherto unrecognized structural alterations in penetrating venules, demonstrates their functional significance and sheds light on the complexity of the relationship between vascular network structure and function in Alzheimer's disease.

Suprachiasmatic vasopressin and the circadian regulation of voluntary locomotor behavior.

A role for arginine vasopressin in the circadian regulation of voluntary locomotor behavior (wheel running activity) was investigated in the golden hamster, Mesocricetus auratus. Spontaneous nocturnal running was suppressed in a dose-dependent manner by systemic injections of vasopressin, and also in a concentration-dependent manner by microinjections directly into the hypothalamic suprachiasmatic nucleus. Pre-injections of a vasopressin V1 receptor antagonist into the nucleus reduced the suppression of behavior by vasopressin. Ethogram analyses revealed that peripheral drug injections predominantly increased grooming, flank marking, and sleep-related behaviors. Central injections did not induce sleep, but increased grooming and periods of 'quiet vigilance' (awake but not moving). Nocturnal behavioral profiles following either peripheral or central injections were similar to those shown by untreated animals in the hour prior to the onset of nocturnal wheel running. Site control vasopressin injections into the medial preoptic area or periaqueductal gray increased flank marking and grooming, but had no significant effect on locomotion, suggesting behavioral specificity of a vasopressin target near the suprachiasmatic nucleus. Both peripheral and central administration increased FOS-like immunoreactivity in the retinorecipient core of the suprachiasmatic nucleus. The distribution of FOS-positive cells overlapped the calbindin subregion, but was more extensive, and most calbindin-positive cells did not co-express FOS. We propose a model of temporal behavioral regulation wherein voluntary behavior, such as nocturnal locomotor activity, is inhibited by the activity of neurons in the suprachiasmatic ventrolateral core that project to the posterior hypothalamus and are driven by rhythmic vasopressin input from the dorsomedial shell.

Micro-ultrasound based characterization of cerebrovasculature following focal ischemic stroke and upon short-term rehabilitation.

Notwithstanding recanalization treatments in the acute stage of stroke, many survivors suffer long-term impairments. Physical rehabilitation is the only widely available strategy for chronic-stage recovery, but its optimization is hindered by limited understanding of its effects on brain structure and function. Using micro-ultrasound, behavioral testing, and electrophysiology, we investigated the impact of skilled reaching rehabilitation on cerebral hemodynamics, motor function, and neuronal activity in a rat model of focal ischemic stroke. A 50 MHz micro-ultrasound transducer and intracortical electrophysiology were utilized to characterize neurovascular changes three weeks following focal ischemia elicited by endothelin-1 injection into the sensorimotor cortex. Sprague-Dawley rats were rehabilitated through tray reaching, and their fine skilled reaching was assessed via the Montoya staircase. Focal ischemia led to a sustained deficit in forelimb reaching; and increased tortuosity of the penetrating vessels in the perilesional cortex; with no lateralization of spontaneous neuronal activity. Rehabilitation improved skilled reaching; decreased cortical vascularity; was associated with elevated peri- vs. contralesional hypercapnia-induced flow homogenization and increased perilesional spontaneous cortical neuronal activity. Our study demonstrated neurovascular plasticity accompanying rehabilitation-elicited functional recovery in the subacute stage following stroke, and multiple micro-ultrasound-based markers of cerebrovascular structure and function modified in recovery from ischemia and upon rehabilitation.

Obesity Facilitates Sex-Specific Improvement In Cognition And Neuronal Function In A Rat Model Of Alzheimer's Disease.

Obesity reduces or increases the risk of developing Alzheimer's disease (AD) depending on whether it is assessed in mid-life or late-life. There is currently no consensus on the relationship between obesity and AD or the mechanism or their interaction. Here, we aim to differentiate the cause-and-effect relationship between obesity and AD in a controlled rat model of AD. We induced obesity in 9-month-old TgF344-AD rats, that is pathology-load wise similar to early symptomatic phase of human AD. To more accurately model human obesity, we fed both TgF344-AD and non-transgenic littermates a varied high-carbohydrate-high-fat diet consisting of human food for 3 months. Obesity increased overall glucose metabolism and slowed cognitive decline in TgF344-AD rats, specifically executive function, without affecting non-transgenic rats. Pathological analyses of prefrontal cortex and hippocampus showed that obesity in TgF344-AD rats produced varied effects, with increased density of myelin and oligodendrocytes, lowered density and activation of microglia that we propose contributes to the cognitive improvement. However, obesity also decreased neuronal density, and promoted deposition of amyloid-beta plaques and tau inclusions. After 6 months on the high-carbohydrate-high-fat diet, detrimental effects on density of neurons, amyloid-beta plaques, and tau inclusions persisted while the beneficial effects on myelin, microglia, and cognitive functions remained albeit with a lower effect size. By examining the effect of sex, we found that both beneficial and detrimental effects of obesity were stronger in female TgF344-AD rats indicating that obesity during early symptomatic phase of AD is protective in females.

Early alterations in brain glucose metabolism and vascular function in a transgenic rat model of Alzheimer's disease.

Alteration in brain metabolism predates clinical onset of Alzheimer's Disease (AD). Realizing its potential as an early diagnostic marker, however, requires understanding how early AD metabolic dysregulation manifests on non-invasive brain imaging. We presently utilized magnetic resonance imaging and spectroscopy to map glucose and ketone metabolic profiles and image cerebrovascular function in a rat model of early stage AD - 9-month-old TgF344-AD (TgAD) rats - and their age-matched non-transgenic (nTg) littermates. Compared to the nTg rats, TgAD rats displayed attenuation in global cerebral and hippocampal vasoreactivity to hypercapnia, by 49 ± 17% and 58 ± 19%, respectively, while their functional hyperemia to somatosensory stimulation diminished by 69 ± 5%. To assess brain glucose uptake, rats were fasted overnight and then challenged with an intravenous infusion of 2-deoxy-D-glucose (2DG). Compared to their non-transgenic littermates, TgAD rats exhibited 99 ± 10% and 52 ± 5% smaller glucose uptake in the entorhinal cortex and the hippocampus, respectively. Moreover, hippocampal glucose uptake reduction in male TgAD rats compared to the nTg was 54 ± 36% greater than the reduction seen in female TgAD rats. TgAD rats also showed a 59 ± 42% increase in total choline level in the hippocampus, suggesting increased membrane turnover. In combination with our earlier findings of impaired electrophysiological metrics at this early stage of AD pathology progression, our findings suggest that subtle neuronal function alterations that would be difficult to assess in a clinical population may be accompanied by MRI-detectable changes in brain glucose metabolism and cerebrovascular function.

Frequency selective neuronal modulation triggers spreading depolarizations in the rat endothelin-1 model of stroke.

Ischemia is one of the most common causes of acquired brain injury. Central to its noxious sequelae are spreading depolarizations (SDs), waves of persistent depolarizations which start at the location of the flow obstruction and expand outwards leading to excitotoxic damage. The majority of acute stage of stroke studies to date have focused on the phenomenology of SDs and their association with brain damage. In the current work, we investigated the role of peri-injection zone pyramidal neurons in triggering SDs by optogenetic stimulation in an endothelin-1 rat model of focal ischemia. Our concurrent two photon fluorescence microscopy data and local field potential recordings indicated that a ≥ 60% drop in cortical arteriolar red blood cell velocity was associated with SDs at the ET-1 injection site. SDs were also observed in the peri-injection zone, which subsequently exhibited elevated neuronal activity in the low-frequency bands. Critically, SDs were triggered by low- but not high-frequency optogenetic stimulation of peri-injection zone pyramidal neurons. Our findings depict a complex etiology of SDs post focal ischemia and reveal that effects of neuronal modulation exhibit spectral and spatial selectivity.

Ciliary neurotrophic factor recruitment of glucagon-like peptide-1 mediates neurogenesis, allowing immortalization of adult murine hypothalamic neurons.

The distinct lack of cell lines derived from the adult brain is evident. Ciliary neurotrophic factor (CNTF) triggers neurogenesis in primary culture from adult mouse hypothalamus, as detected by bromodeoxyuridine and Ki67 immunostaining. Using SV-40 T-antigen, we immortalized dividing neurons and generated clonal cell lines expressing neuropeptides and receptors involved in neuroendocrine function. We hypothesized that proglucagon-derived peptides may be the mechanistic downstream effectors of CNTF due to documented neuroprotective and proliferative effects. Indeed, proglucagon gene expression was induced by CNTF, and exposure of primary cells to glucagon-like peptide-1 receptor (GLP-1) agonist, exendin-4, induced cell proliferation. Intracerebroventricular injection of CNTF into adult mice caused increased expression of proglucagon peptide in the hypothalamus. Using a specific GLP-1-receptor antagonist, we found that neurogenesis was significantly attenuated and primary culture from GLP-1-receptor-knockout mice lacked CNTF-mediated neuronal proliferation, thus linking the induction of neurogenesis in the hypothalamus to GLP-1-receptor signaling.

Saturation transfer properties of tumour xenografts derived from prostate cancer cell lines 22Rv1 and DU145.

Histopathology is currently the most reliable tool in assessing the aggressiveness and prognosis of solid tumours. However, developing non-invasive modalities for tumour evaluation remains crucial due to the side effects and complications caused by biopsy procedures. In this study, saturation transfer MRI was used to investigate the microstructural and metabolic properties of tumour xenografts in mice derived from the prostate cancer cell lines 22Rv1 and DU145, which express different aggressiveness. The magnetization transfer (MT) and chemical exchange saturation transfer (CEST) effects, which are associated with the microstructural and metabolic properties in biological tissue, respectively, were analyzed quantitatively and compared amongst different tumour types and regions. Histopathological staining was performed as a reference. Higher cellular density and metabolism expressed in more aggressive tumours (22Rv1) were associated with larger MT and CEST effects. High collagen content in the necrotic regions might explain their higher MT effects compared to tumour regions.

An Automated Segmentation Pipeline for Intratumoural Regions in Animal Xenografts Using Machine Learning and Saturation Transfer MRI.

Saturation transfer MRI can be useful in the characterization of different tumour types. It is sensitive to tumour metabolism, microstructure, and microenvironment. This study aimed to use saturation transfer to differentiate between intratumoural regions, demarcate tumour boundaries, and reduce data acquisition times by identifying the imaging scheme with the most impact on segmentation accuracy. Saturation transfer-weighted images were acquired over a wide range of saturation amplitudes and frequency offsets along with T1 and T2 maps for 34 tumour xenografts in mice. Independent component analysis and Gaussian mixture modelling were used to segment the images and identify intratumoural regions. Comparison between the segmented regions and histopathology indicated five distinct clusters: three corresponding to intratumoural regions (active tumour, necrosis/apoptosis, and blood/edema) and two extratumoural (muscle and a mix of muscle and connective tissue). The fraction of tumour voxels segmented as necrosis/apoptosis quantitatively matched those calculated from TUNEL histopathological assays. An optimal protocol was identified providing reasonable qualitative agreement between MRI and histopathology and consisting of T1 and T2 maps and 22 magnetization transfer (MT)-weighted images. A three-image subset was identified that resulted in a greater than 90% match in positive and negative predictive value of tumour voxels compared to those found using the entire 24-image dataset. The proposed algorithm can potentially be used to develop a robust intratumoural segmentation method.

Refinement of a chronic cranial window implant in the rat for longitudinal in vivo two-photon fluorescence microscopy of neurovascular function.

Longitudinal studies using two-photon fluorescence microscopy (TPFM) are critical for facilitating cellular scale imaging of brain morphology and function. Studies have been conducted in the mouse due to their relatively higher transparency and long term patency of a chronic cranial window. Increasing availability of transgenic rat models, and the range of established behavioural paradigms, necessitates development of a chronic preparation for the rat. However, surgical craniotomies in the rat present challenges due to craniotomy closure by wound healing and diminished image quality due to inflammation, restricting most rat TPFM experiments to acute preparations. Long-term patency is enabled by employing sterile surgical technique, minimization of trauma with precise tissue handling during surgery, judicious selection of the size and placement of the craniotomy, diligent monitoring of animal physiology and support throughout the surgery, and modification of the home cage for long-term preservation of cranial implants. Immunohistochemical analysis employing the glial fibrillary acidic protein (GFAP) and ionized calcium-binding adaptor molecule-1 (Iba-1) showed activation and recruitment of astrocytes and microglia/macrophages directly inferior to the cranial window at one week after surgery, with more diffuse response in deeper cortical layers at two weeks, and amelioration around four weeks post craniotomy. TPFM was conducted up to 14 weeks post craniotomy, reaching cortical depths of 400 µm to 600 µm at most time-points. The rate of signal decay with increasing depth and maximum cortical depth attained had greater variation between individual rats at a single time-point than within a rat across time.

Combinatorial Treatment Using Umbilical Cord Perivascular Cells and Aß Clearance Rescues Vascular Function Following Transient Hypertension in a Rat Model of Alzheimer Disease.

Transient hypertension is a risk factor for Alzheimer disease (AD), but the effects of this interaction on brain vasculature are understudied. Addressing vascular pathology is a promising avenue to potentiate the efficacy of treatments for AD. We used arterial spin labeling magnetic resonance imaging to longitudinally assess brain vascular function and immunohistopathology to examine cerebrovascular remodeling and amyloid load. Hypertension was induced for 1 month by administration of l-NG-nitroarginine-methyl-ester in TgF344-AD rats at the prodromal stage. Following hypertension, nontransgenic rats showed transient cerebrovascular changes, whereas TgF344-AD animals exhibited sustained alterations in cerebrovascular function. Human umbilical cord perivascular cells in combination with scyllo-inositol, an inhibitor of Aß oligomerization, resulted in normalization of hippocampal vascular function and remodeling, in contrast to either treatment alone. Prodromal stage hypertension exacerbates latter AD pathology, and the combination of human umbilical cord perivascular cells with amyloid clearance promotes cerebrovascular functional recovery.

Oophorectomy Reduces Estradiol Levels and Long-Term Spontaneous Neurovascular Recovery in a Female Rat Model of Focal Ischemic Stroke.

Although epidemiological evidence suggests significant sex and gender-based differences in stroke risk and recovery, females have been widely under-represented in preclinical stroke research. The neurovascular sequelae of brain ischemia in females, in particular, are largely uncertain. We set out to address this gap by a multimodal in vivo study of neurovascular recovery from endothelin-1 model of cortical focal-stroke in sham vs. ovariectomized female rats. Three weeks post ischemic insult, sham operated females recapitulated the phenotype previously reported in male rats in this model, of normalized resting perfusion but sustained peri-lesional cerebrovascular hyperreactivity. In contrast, ovariectomized (Ovx) females showed reduced peri-lesional resting blood flow, and elevated cerebrovascular responsivity to hypercapnia in the peri-lesional and contra-lateral cortices. Electrophysiological recordings showed an attenuation of theta to low-gamma phase-amplitude coupling in the peri-lesional tissue of Ovx animals, despite relative preservation of neuronal power. Further, this chronic stage neuronal network dysfunction was inversely correlated with serum estradiol concentration. Our pioneering data demonstrate dramatic differences in spontaneous recovery in the neurovascular unit between Ovx and Sham females in the chronic stage of stroke, underscoring the importance of considering hormonal-dependent aspects of the ischemic sequelae in the development of novel therapeutic approaches and patient recruitment in clinical trials.

Differentiation of Normal and Radioresistant Prostate Cancer Xenografts Using Magnetization Transfer-Prepared MRI.

The ability of MRI to differentiate between normal and radioresistant cancer was investigated in prostate tumour xenografts in mice. Specifically, the process of magnetization exchange between water and other molecules was studied. It was found that magnetization transfer from semisolid macromolecules (MT) and chemical exchange saturation transfer (CEST) combined were significantly different between groups (p < 0.01). Further, the T2 relaxation of the semisolid macromolecular pool (T2,B), a parameter specific to MT, was found to be significantly different (p < 0.01). Also significantly different were the rNOE contributions associated with methine groups at -0.9 ppm with a saturation B1 of 0.5 µT (p < 0.01) and with other aliphatic groups at -3.3 ppm with 0.5 and 2 µT (both p < 0.05). Independently, using a live-cell metabolic assay, normal cells were found to have a greater metabolic rate than radioresistant ones. Thus, MRI provides a novel, in vivo method to quantify the metabolic rate of tumours and predict their radiosensitivity.

3D morphological analysis of the mouse cerebral vasculature: Comparison of in vivo and ex vivo methods.

Ex vivo 2-photon fluorescence microscopy (2PFM) with optical clearing enables vascular imaging deep into tissue. However, optical clearing may also produce spherical aberrations if the objective lens is not index-matched to the clearing material, while the perfusion, clearing, and fixation procedure may alter vascular morphology. We compared in vivo and ex vivo 2PFM in mice, focusing on apparent differences in microvascular signal and morphology. Following in vivo imaging, the mice (four total) were perfused with a fluorescent gel and their brains fructose-cleared. The brain regions imaged in vivo were imaged ex vivo. Vessels were segmented in both images using an automated tracing algorithm that accounts for the spatially varying PSF in the ex vivo images. This spatial variance is induced by spherical aberrations caused by imaging fructose-cleared tissue with a water-immersion objective. Alignment of the ex vivo image to the in vivo image through a non-linear warping algorithm enabled comparison of apparent vessel diameter, as well as differences in signal. Shrinkage varied as a function of diameter, with capillaries rendered smaller ex vivo by 13%, while penetrating vessels shrunk by 34%. The pial vasculature attenuated in vivo microvascular signal by 40% 300 µm below the tissue surface, but this effect was absent ex vivo. On the whole, ex vivo imaging was found to be valuable for studying deep cortical vasculature.

Effects of voluntary exercise on structure and function of cortical microvasculature.

Aerobic activity has been shown highly beneficial to brain health, yet much uncertainty still surrounds the effects of exercise on the functioning of cerebral microvasculature. This study used two-photon fluorescence microscopy to examine cerebral hemodynamic alterations as well as accompanying geometric changes in the cortical microvascular network following five weeks of voluntary exercise in transgenic mice endogenously expressing tdTomato in vascular endothelial cells to allow visualization of microvessels irrespective of their perfusion levels. We found a diminished microvascular response to a hypercapnic challenge (10% FiCO2) in running mice when compared to that in nonrunning controls despite commensurate increases in transcutaneous CO2 tension. The flow increase to hypercapnia in runners was 70% lower than that in nonrunners (p = 0.0070) and the runners' arteriolar red blood cell speed changed by only half the amount seen in nonrunners (p = 0.0085). No changes were seen in resting hemodynamics or in the systemic physiological parameters measured. Although a few unperfused new vessels were observed on visual inspection, running did not produce significant morphological differences in the microvascular morphometric parameters, quantified following semiautomated tracking of the microvascular networks. We propose that voluntary running led to increased cortical microvascular efficiency and desensitization to CO2 elevation.