Is CAA a perivascular brain clearance disease? A discussion of the evidence to date and outlook for future studies.

The brain's network of perivascular channels for clearance of excess fluids and waste plays a critical role in the pathogenesis of several neurodegenerative diseases including cerebral amyloid angiopathy (CAA). CAA is the main cause of hemorrhagic stroke in the elderly, the most common vascular comorbidity in Alzheimer's disease and also implicated in adverse events related to anti-amyloid immunotherapy. Remarkably, the mechanisms governing perivascular clearance of soluble amyloid ß-a key culprit in CAA-from the brain to draining lymphatics and systemic circulation remains poorly understood. This knowledge gap is critically important to bridge for understanding the pathophysiology of CAA and accelerate development of targeted therapeutics. The authors of this review recently converged their diverse expertise in the field of perivascular physiology to specifically address this problem within the framework of a Leducq Foundation Transatlantic Network of Excellence on Brain Clearance. This review discusses the overarching goal of the consortium and explores the evidence supporting or refuting the role of impaired perivascular clearance in the pathophysiology of CAA with a focus on translating observations from rodents to humans. We also discuss the anatomical features of perivascular channels as well as the biophysical characteristics of fluid and solute transport.

Subminute thermal damage to cell types present in the skin.

INTRODUCTION: Psoriasis is characterized by an increase in the proliferation of keratinocytes and nerve fiber activity, contributing to the typical skin lesions. Pulsed Dye Laser (PDL) treatment is effective for the treatment of psoriatic lesions but its mechanism remains unclear. One hypothesis is that PDL causes thermal damage by the diffusion of heat to neighboring structures in lesional skin. There is limited information on the thermal sensitivity of these neighboring skin cells when exposed to hyperthermia for durations lasting less than a minute. Our study aimed to investigate the cell-specific responses to heat using sub-minute exposure times and moderate to ablative hyperthermia. MATERIALS AND METHODS: Cultured human endothelial cells, smooth muscle cells, neuronal cells, and keratinocytes were exposed to various time (2-20 sec) and temperature (45-70 °C) combinations. Cell viability was assessed by measuring intracellular ATP content 24 h after thermal exposure and this data was used to calculate fit parameters for the Arrhenius model and CEM43 calculations. RESULTS: Our results show significant differences in cell survival between cell types (p < 0.0001). Especially within the range of 50-60 °C, survival of neuronal cells and keratinocytes was significantly less than that of endothelial and smooth muscle cells. No statistically significant difference was found in the lethal dose (LT50) of thermal energy between neuronal cells and keratinocytes. However, CEM43 calculations showed significant differences between all four cell types. CONCLUSION: The results imply that there is a cell-type-dependent sensitivity to thermal damage which suggests that neuronal cells and keratinocytes are particularly susceptible to diffusing heat from laser treatment. Damage to these cells may aid in modulating the neuro-inflammatory pathways in psoriasis. These data provide insight into the potential mechanisms of PDL therapy for psoriasis and advance our understanding of how thermal effects may play a role in its effectiveness.

Current Understanding of the Anatomy, Physiology, and Magnetic Resonance Imaging of Neurofluids: Update From the 2022 "ISMRM Imaging Neurofluids Study group" Workshop in Rome.

Neurofluids is a term introduced to define all fluids in the brain and spine such as blood, cerebrospinal fluid, and interstitial fluid. Neuroscientists in the past millennium have steadily identified the several different fluid environments in the brain and spine that interact in a synchronized harmonious manner to assure a healthy microenvironment required for optimal neuroglial function. Neuroanatomists and biochemists have provided an incredible wealth of evidence revealing the anatomy of perivascular spaces, meninges and glia and their role in drainage of neuronal waste products. Human studies have been limited due to the restricted availability of noninvasive imaging modalities that can provide a high spatiotemporal depiction of the brain neurofluids. Therefore, animal studies have been key in advancing our knowledge of the temporal and spatial dynamics of fluids, for example, by injecting tracers with different molecular weights. Such studies have sparked interest to identify possible disruptions to neurofluids dynamics in human diseases such as small vessel disease, cerebral amyloid angiopathy, and dementia. However, key differences between rodent and human physiology should be considered when extrapolating these findings to understand the human brain. An increasing armamentarium of noninvasive MRI techniques is being built to identify markers of altered drainage pathways. During the three-day workshop organized by the International Society of Magnetic Resonance in Medicine that was held in Rome in September 2022, several of these concepts were discussed by a distinguished international faculty to lay the basis of what is known and where we still lack evidence. We envision that in the next decade, MRI will allow imaging of the physiology of neurofluid dynamics and drainage pathways in the human brain to identify true pathological processes underlying disease and to discover new avenues for early diagnoses and treatments including drug delivery. Evidence level: 1 Technical Efficacy: Stage 3.

Cerebrospinal fluid turnover as a driver of brain clearance.

Cerebrospinal fluid (CSF) has historically been considered to function as a sink for brain-derived waste disposal. Recent work suggested that CSF interacts even more intensely with brain tissue than previously recognized, through perivascular spaces that penetrate the brain. Cardiac pulsations, vasomotion, and respiration have been suggested to drive CSF flow in these perivascular spaces, thereby enhancing waste clearance. However, the intrinsic role of CSF production in relation to its distribution volume (turnover) is not an explicit component of recent concepts on brain clearance. Here, we review the work on CSF turnover and volume, focusing on preclinical evidence. Herein, we highlight the use of MRI in establishing CSF-related parameters. We describe the impact of sleep, effect of anesthesia, aging, and hypertension on CSF turnover, and how this relates to brain clearance. Evaluation of the available evidence suggests that CSF turnover is a major determinant in brain clearance. In addition, we propose that several putative drivers of brain clearance, but also conditions associated with impaired clearance, such as aging, may actually relate to altered CSF turnover.

Effects of pulsed dye laser treatment in psoriasis: A nerve-wrecking process?

Pulsed dye laser (PDL) therapy can be effective in treating psoriasis, with a long duration of remission. Although PDL therapy, albeit on a modest scale, is being used for decades now, the underlying mechanisms responsible for the long-term remission of psoriasis remain poorly understood. The selective and rapid absorption of energy by the blood causes heating of the vascular wall and surrounding structures, like perivascular nerves. Several studies indicate the importance of nerves in psoriatic inflammation. Interestingly, denervation leads to a spontaneous remission of the psoriatic lesion. Among all dermal nerves, the perivascular nerves are the most likely to be affected during PDL treatment, possibly impairing the neuro-inflammatory processes that promote T-cell activation, expression of adhesion molecules, leukocyte infiltration and cytokine production. Repeated PDL therapy could cause a prolonged loss of innervation through nerve damage, or result in a 'reset' of neurogenic inflammation after temporary denervation. The current hypothesis provides strong arguments that PDL treatment affects nerve fibres in the skin and thereby abrogates the persistent and exaggerated inflammatory process underlying psoriasis, causing a long-term remission of psoriasis.

Effect of long-term antihypertensive treatment on cerebrovascular structure and function in hypertensive rats.

Midlife hypertension is an important risk factor for cognitive impairment and dementia, including Alzheimer's disease. We investigated the effects of long-term treatment with two classes of antihypertensive drugs to determine whether diverging mechanisms of blood pressure lowering impact the brain differently. Spontaneously hypertensive rats (SHR) were either left untreated or treated with a calcium channel blocker (amlodipine) or beta blocker (atenolol) until one year of age. The normotensive Wistar Kyoto rat (WKY) was used as a reference group. Both drugs lowered blood pressure equally, while only atenolol decreased heart rate. Cerebrovascular resistance was increased in SHR, which was prevented by amlodipine but not atenolol. SHR showed a larger carotid artery diameter with impaired pulsatility, which was prevented by atenolol. Cerebral arteries demonstrated inward remodelling, stiffening and endothelial dysfunction in SHR. Both treatments similarly improved these parameters. MRI revealed that SHR have smaller brains with enlarged ventricles. In addition, neurofilament light levels were increased in cerebrospinal fluid of SHR. However, neither treatment affected these parameters. In conclusion, amlodipine and atenolol both lower blood pressure, but elicit a different hemodynamic profile. Both medications improve cerebral artery structure and function, but neither drug prevented indices of brain damage in this model of hypertension.

Extravasation of biodegradable microspheres in the rat brain.

Drug development for neurological diseases is greatly impeded by the presence of the blood-brain barrier (BBB). We and others previously reported on extravasation of micrometer-sized particles from the cerebral microcirculation - across the BBB - into the brain tissue over the course of several weeks. This mechanism could potentially be used for sustained parenchymal drug delivery after extravasation of biodegradable microspheres. As a first step toward this goal, we set out to evaluate the extravasation potential in the rat brain of three classes of biodegradable microspheres with drug-carrying potential, having a median diameter of 13 µm (80% within 8-18 µm) and polyethylene glycol concentrations of 0%, 24% and 36%. Extravasation, capillary recanalization and tissue damage were determined in a rat cerebral microembolization model at day 14 after microsphere injection. Microspheres of all three classes had the potential to extravasate from the vessel into the brain parenchyma, with microspheres without polyethylene glycol extravasating the fastest. Microembolization with biodegradable microspheres led to impaired local capillary perfusion, which was substantially restored after bead extravasation. We did not observe overt tissue damage after microembolization with any microsphere: we found very limited BBB disruption (IgG extravasation), no microgliosis (Iba1 staining) and no large neuronal infarctions (NeuN staining). In conclusion, biodegradable microspheres with different polymer compositions can extravasate into the brain parenchyma while causing minimal tissue damage.

Perivascular clearance of blood proteins after blood-brain barrier disruption in a rat model of microinfarcts.

Microinfarcts result in a transient loss of the blood-brain barrier (BBB) in the ischemic territory. This leads to the extravasation of blood proteins into the brain parenchyma. It is not clear how these proteins are removed. Here we studied the role of perivascular spaces in brain clearance from extravasated blood proteins. Male and female Wistar rats were infused with microspheres of either 15, 25, or 50 µm in diameter (n = 6 rats per group) via the left carotid artery. We infused either 25,000 microspheres of 15 µm, 5500 of 25 µm, or 1000 of 50 µm. One day later, rats were infused with lectin and hypoxyprobe to label perfused blood vessels and hypoxic areas, respectively. Rats were then euthanized and perfusion-fixed. Brains were excised, sectioned, and analyzed using immunostaining and confocal imaging. Microspheres induced a size-dependent increase in ischemic volume per territory, but the cumulative ischemic volume was similar in all groups. The total volumes of ischemia, hypoxia and infarction affected 1-2 % of the left hemisphere. Immunoglobulins (IgG) were present in ischemic brain tissue surrounding lodged microspheres in all groups. In addition, staining for IgG was found in perivascular spaces of blood vessels nearby areas of BBB disruption. About 2/3 of these vessels were arteries, while the remaining 1/3 of these vessels were veins. The subarachnoid space (SAS) of the affected hemisphere stained stronger for IgG than the contralateral hemisphere in all groups: +27 %, +44 % and +27 % respectively. Microspheres of various sizes induce a local loss of BBB integrity, evidenced by parenchymal IgG staining. The presence of IgG in perivascular spaces of both arteries and veins distinct from the ischemic territories suggests that both contribute to the removal of blood proteins. The strong staining for IgG in the SAS of the affected hemisphere suggests that this perivascular route egresses via the CSF. Perivascular spaces therefore play a previously unrecognized role in tissue clearance of fluid and extravasated proteins after BBB disruption induced by microinfarcts.

Mechanistic and therapeutic role of NLRP3 inflammasome in the pathogenesis of Alzheimer's disease.

Alzheimer's disease (AD), a progressive neurodegenerative disorder, has emerged as the most common form of dementia in the elderly. Several pathological hallmarks have been identified, including neuroinflammation. A comprehensive insight into the underlying mechanisms that can fuel the development of novel therapeutic approaches is necessary because of the alarmingly rapid increase in the frequency of incidence. Recently, NLRP3 inflammasome was identified as a critical mediator of neuroinflammation. Activation of nucleotide-binding domain (NOD)-like receptor protein 3 (NLRP3) inflammasome by amyloid, neurofibrillary tangles, impaired autophagy and endoplasmic reticulum stress, triggers the release of pro-inflammatory cytokines such as IL-1ß and IL-18. Subsequently, these cytokines can promote neurodegeneration and cognitive impairment. It is well established that genetic or pharmacological ablation of NLRP3 alleviates AD-related pathological features in in vitro and in vivo models. Therefore, several synthetic and natural compounds have been identified that exhibit the potential to inhibit NLRP3 inflammasome and alleviate AD-associated pathology. The current review article will highlight the various mechanisms by which activation of NLRP3 inflammation occurs during Alzheimer's disease, and how it influences neuroinflammation, neurodegeneration and cognitive impairment. Moreover, we will summarise the different small molecules that possess the potential to inhibit NLRP3 and can pave the path for developing novel therapeutic interventions for AD.

Physiology of Glymphatic Solute Transport and Waste Clearance from the Brain.

This review focuses on the physiology of glymphatic solute transport and waste clearance, using evidence from experimental animal models as well as from human studies. Specific topics addressed include the biophysical characteristics of fluid and solute transport in the central nervous system, glymphatic-lymphatic coupling, as well as the role of cerebrospinal fluid movement for brain waste clearance. We also discuss the current understanding of mechanisms underlying increased waste clearance during sleep.

Quantitative 3D analysis of tissue damage in a rat model of microembolization.

There is a discrepancy between successful recanalization and good clinical outcome after endovascular treatment (EVT) in acute ischemic stroke patients. During removal of a thrombus, a shower of microemboli may release and lodge to the distal circulation. The objective of this study was to determine the extent of damage on brain tissue caused by microemboli. In a rat model of microembolization, a mixture of microsphere (MS) sizes (15, 25 and 50 µm diameter) was injected via the left internal carotid artery. A 3D image of the left hemisphere was reconstructed and a point-pattern spatial analysis was applied based on G- and K-functions to unravel the spatial correlation between MS and the induced hypoxia or infarction. We show a spatial correlation between MS and hypoxia or infarction spreading up to a distance of 1000-1500 µm. These results imply that microemboli, which individually may not always be harmful, can interact and result in local areas of hypoxia or even infarction when lodged in large numbers.

Brain solute transport is more rapid in periarterial than perivenous spaces.

Fluid flow in perivascular spaces is recognized as a key component underlying brain transport and clearance. An important open question is how and to what extent differences in vessel type or geometry affect perivascular fluid flow and transport. Using computational modelling in both idealized and image-based geometries, we study and compare fluid flow and solute transport in pial (surface) periarterial and perivenous spaces. Our findings demonstrate that differences in geometry between arterial and venous pial perivascular spaces (PVSs) lead to higher net CSF flow, more rapid tracer transport and earlier arrival times of injected tracers in periarterial spaces compared to perivenous spaces. These findings can explain the experimentally observed rapid appearance of tracers around arteries, and the delayed appearance around veins without the need of a circulation through the parenchyma, but rather by direct transport along the PVSs.

Mapping Solute Clearance From the Mouse Hippocampus Using a 3D Imaging Cryomicrotome.

The hippocampus is susceptible to protein aggregation in neurodegenerative diseases such as Alzheimer's disease. This protein accumulation is partially attributed to an impaired clearance; however, the removal pathways for fluids and waste products are not fully understood. The aim of this study was therefore to map the clearance pathways from the mouse brain. A mixture of two fluorescently labeled tracers with different molecular weights was infused into the hippocampus. A small subset of mice (n = 3) was sacrificed directly after an infusion period of 10 min to determine dispersion of the tracer due to the infusion, while another group was sacrificed after spreading of the tracers for an additional 80 min (n = 7). Upon sacrifice, mice were frozen and sectioned as a whole by the use of a custom-built automated imaging cryomicrotome. Detailed 3D reconstructions were created to map the tracer spreading. We observed that tracers distributed over the hippocampus and entered adjacent brain structures, such as the cortex and cerebroventricular system. An important clearance pathway was found along the ventral part of the hippocampus and its bordering interpeduncular cistern. From there, tracers left the brain via the subarachnoid spaces in the directions of both the nose and the spinal cord. Although both tracers followed the same route, the small tracer distributed further, implying a major role for diffusion in addition to convection. Taken together, these results reveal an important clearance pathway of solutes from the hippocampus.

Recovery of Hypoxic Regions in a Rat Model of Microembolism.

OBJECTIVES: Endovascular treatment (EVT) has become the standard of care for acute ischemic stroke. Despite successful recanalization, a limited subset of patients benefits from the new treatment. Human MRI studies have shown that during removal of the thrombus, a shower of microclots is released from the initial thrombus, possibly causing new ischemic lesions. The aim of the current study is to quantify tissue damage following microembolism. MATERIALS AND METHODS: In a rat model, microembolism was generated by injection of a mixture of polystyrene fluorescent microspheres (15, 25 and 50 µm in diameter). The animals were killed at three time-points: day 1, 3 or 7. AMIRA and IMARIS software was used for 3D reconstruction of brain structure and damage, respectively. CONCLUSIONS: Microembolism induces ischemia, hypoxia and infarction. Infarcted areas persist, but hypoxic regions recover over time suggesting that repair processes in the brain rescue the regions at risk.

Microembolus clearance through angiophagy is an auxiliary mechanism preserving tissue perfusion in the rat brain.

Considering its intolerance to ischemia, it is of critical importance for the brain to efficiently process microvascular occlusions and maintain tissue perfusion. In addition to collateral microvascular flow and enzymatic degradation of emboli, the endothelium has the potential to engulf microparticles and thereby recanalize the vessel, through a process called angiophagy. Here, we set out to study the dynamics of angiophagy in relation to cytoskeletal remodeling in vitro and reperfusion in vivo. We show that polystyrene microspheres and fibrin clots are actively taken up by (brain) endothelial cells in vitro, and chart the dynamics of the actin cytoskeleton during this process using live cell imaging. Whereas microspheres were taken up through the formation of a cup structure by the apical endothelial membrane, fibrin clots were completely engulfed by the cells, marked by dense F-actin accumulation surrounding the clot. Both microspheres and fibrin clots were retained in the endothelial cells. Notably, fibrin clots were not degraded intracellularly. Using an in vivo microembolization rat model, in which microparticles are injected into the common carotid artery, we found that microspheres are transported by the endothelium from the microvasculature into the brain parenchyma. Microembolization with microspheres caused temporal opening of the blood-brain barrier and vascular nonperfusion, followed by microsphere extravasation and restoration of vessel perfusion over time. Taken together, angiophagy is accompanied by active cytoskeletal remodeling of the endothelium, and is an effective mechanism to restore perfusion of the occluded microvasculature in vivo.

Altered brain fluid management in a rat model of arterial hypertension.

BACKGROUND: Proper neuronal function is directly dependent on the composition, turnover, and amount of interstitial fluid that bathes the cells. Most of the interstitial fluid is likely to be derived from ion and water transport across the brain capillary endothelium, a process that may be altered in hypertension due to vascular pathologies as endothelial dysfunction and arterial remodelling. In the current study, we investigated the effects of hypertension on the brain for differences in the water homeostasis. METHODS: Magnetic resonance imaging (MRI) was performed on a 7T small animal MRI system on male spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY) of 10 months of age. The MRI protocol consisted of T2-weighted scans followed by quantitative apparent diffusion coefficient (ADC) mapping to measure volumes of different anatomical structures and water diffusion respectively. After MRI, we assessed the spatial distribution of aquaporin 4 expression around blood vessels. RESULTS: MRI analysis revealed a significant reduction in overall brain volume and remarkably higher cerebroventricular volume in SHR compared to WKY. Whole brain ADC, as well as ADC values of a number of specific anatomical structures, were significantly lower in hypertensive animals. Additionally, SHR exhibited higher brain parenchymal water content. Immunohistochemical analysis showed a profound expression of aquaporin 4 around blood vessels in both groups, with a significantly larger area of influence around arterioles. Evaluation of specific brain regions revealed a decrease in aquaporin 4 expression around capillaries in the corpus callosum of SHR. CONCLUSION: These results indicate a shift in the brain water homeostasis of adult hypertensive rats.

Celiprolol but not losartan improves the biomechanical integrity of the aorta in a mouse model of vascular Ehlers-Danlos syndrome.

AIMS: Antihypertensive drugs are included in the medical therapy of vascular Ehlers-Danlos syndrome (vEDS). The ß-blocker celiprolol has been suggested to prevent arterial damage in vEDS, but the underlying mechanism remains unclear. It is also unknown whether the widely used angiotensin II receptor type 1 antagonist losartan has a therapeutic effect in vEDS. Here, we evaluated the impact of celiprolol and losartan on the biomechanical integrity of the vEDS thoracic aorta. METHODS AND RESULTS: We established a new approach to measure the maximum tensile force at rupture of uniaxially stretched murine thoracic aortic rings. In a vEDS model, which we (re-)characterized here at molecular level, heterozygous mice showed a significant reduction in the rupture force compared to wild-type mice, reflecting the increased mortality due to aortic rupture. For the assessment of treatment effects, heterozygous mice at 4 weeks of age underwent a 4-week treatment with celiprolol, losartan, and, as a proof-of-concept drug, the matrix metalloproteinase inhibitor doxycycline. Compared to age- and sex-matched untreated heterozygous mice, treatment with doxycycline or celiprolol resulted in a significant increase of rupture force, whereas no significant change was detected upon losartan treatment. CONCLUSIONS: In a vEDS model, celiprolol or doxycycline, but not losartan, can improve the biomechanical integrity of the aortic wall, thereby potentially reducing the risk of dissection and rupture. As doxycycline is a broad-spectrum antibiotic with considerable side effects, celiprolol may be more suitable for a long-term therapy and thus rather indicated for the medication of patients with vEDS.

Extravasation of Microspheres in a Rat Model of Silent Brain Infarcts.

Background and Purpose- We developed a rat model of silent brain infarcts based on microsphere infusion and investigated their impact on perfusion and tissue damage. Second, we studied the extent and mechanisms of perfusion recovery. Methods- At day 0, 15 µm fluorescent microspheres were injected into the right common carotid artery of F344 rats. At days 1, 7, or 28, the brain was removed, cut in 100-µm cryosections, and processed for immunofluorescent staining and analysis. Results- Injection of microspheres caused mild and transient damage to the treated hemisphere, with a decrease in perfused capillary volume at day 1, as compared with the untreated hemisphere. At day 1 but not at days 7 and 28, we observed IgG staining outside of the vessels, indicating vessel leakage. All microspheres were located inside the lumen of the vessels at day 1, whereas the vast majority (≈80%) of the microspheres were extravascular at day 7, and 100% at day 28. This was accompanied by restoration of perfused capillary volume. Conclusions- Microspheres cause mild and transient damage, and effective extravasation mechanisms exist in the brain to clear microsized emboli from the vessels.

Paravascular spaces: entry to or exit from the brain?

NEW FINDINGS: What is the topic of this review? In this symposium report, we review the glymphatic clearance from the brain. What advances does it highlight? Evaluation of the evidence indicates that cerebrospinal fluid flows along paravascular spaces at the surface of the brain. However, bulk flow along penetrating arteries into the brain, followed by exit along veins, requires further confirmation. Clearance from the brain, based on mixing, might provide an alternative explanation for experimental findings. ABSTRACT: The interstitial fluid of the brain provides the environment for proper neuronal function. Maintenance of the volume and composition of interstitial fluid requires regulation of the influx and removal of water, ions, nutritive and waste products. The recently described glymphatic pathway might contribute to some of these functions. It is proposed that cerebrospinal fluid enters the brain via paravascular spaces along arteries, mixes with interstitial fluid, and leaves the brain via paravascular spaces along veins. In this symposium report, we review the glymphatic concept, its concerns, and alternative views on interstitial fluid-cerebrospinal fluid exchange.