The glycocalyx is an important layer lining the internal wall of blood vessels as it can attenuate BBB permeability and is a vasculoprotective [35] barrier between the circulating blood and the endothelium facing the lumen [36,37]. The glycocalyx consists predominantly of proteoglycan and a glycoprotein backbone with glycosaminoglycan connections [35]. It is under constant remodeling due to hemodynamic changes, enzymatic degradation, and shear stress [36]. Interestingly, SAH can disrupt the glycocalyx, often associated with the upregulation of inflammatory cytokine production, reduced nitric oxide production, and neurovascular uncoupling [38].

aSAH can trigger upregulation of pro-inflammatory cytokines (e.g., TNF-alpha, IL-1, IL-6), which can in turn initiate cytokine-induced breakdown of the glycocalyx, leading to endothelium and adhesion molecule (i.e., VCAM, ICAM) dysfunction [38,39,40,41]. While glycocalyx degradation can expose the endothelium to pro-inflammatory cytokine-mediated damage, it also reduces endothelial nitric oxide [38].

During SAH, glycocalyx degradation occurs from the increased expression of inflammatory markers (e.g., IL-1, IL-6, TNF-alpha), atrial natriuretic peptides, and vascular shear stress [37]. Following disruption of the glycocalyx, endothelial NO production is disrupted, leading to vascular smooth muscle cell-mediated vasoconstriction [42]. Additionally, glycocalyx’s breakdown exposes its molecular backbone, which contains the molecules glypican and heparin sulfate, both of which serve to increase neutrophil migration and adhesion and potentiate platelet activation [43,44]. These compounding factors lead to further vascular compromise.

Endothelial dysfunction in aSAH is characterized by the loss of NO production, unregulated coagulation (via coagulation cascade dysfunction), and enhanced permeability due to glycocalyx impairment [45,46]. Loss of the glycocalyx promotes coagulation since anticoagulant molecules (e.g., anti-thrombin, tissue factor inhibitor pathway, and vWF) are decreased to prevent platelet adhesion and aggregation [38]. aSAH-mediated damage to the endothelium can also decrease prostacyclin release, which prevents platelet adhesion [38]. Furthermore, TNF-α stimulates exposed endothelium to upregulate P-selectin and increase platelet–leukocyte–endothelial cell interactions [47,48].

aSAH can lead to infiltration of neutrophils at the site of injury, along with systemic neutrophilia via enhanced IL-6 [54]. This process precipitates neutrophil recruitment to the brain between 12 and 48 h following aSAH [55]. Additionally, microglia activation and monocyte recruitment readily occur within the first 48 h of aSAH to promote neuroinflammation [55]. Neutrophil accumulation on endothelial membranes increases oxidative stress (via myeloperoxidase) and lipid peroxidation, resulting in endothelium damage [53,54,56,57]. Toll-like receptor 4 (TLR4) is enhanced in the activated microglia and macrophages, resulting in the secretion of TNF [58]. Free heme from RBC hemolysis forms reactive oxygen species, resulting in upregulation of metalloproteinase-9 (MMP-9) and breakdown of the vascular basement membrane [59].

Naïve astrocyte activation occurs during SAH, transforming the cells into the A1 neurotoxic phenotype [60,61]. The A1 phenotype is characterized by upregulation of glial fibrillary acidic protein (GFAP), S100 calcium binding protein B (S100B), and C3, H2-D1, and serping1 cell markers [62]. A1 astrocytes are a form of reactive astrocyte, promoting cell death by releasing proinflammatory cytokines [61]. A2 astrocytes are another phenotypic variant in reactive astrocytes that play a neuroprotective role, in contrast to the dominant A1 neurotoxic phenotype found in SAH [63,64,65,66]. Reactive astrocytes (RAs) display a spectrum of pro- and anti-inflammatory profiles, rather than the simple A1 and A2 phenotypes based on genetic sequencing [67]. Overall, the pro-inflammatory RAs found in SAH exhibit strong neurotoxicity—forming fewer synapses and killing neurons with detached axons [68,69]. Activated astrocytes also undergo morphological changes after SAH, such as end feet swelling and protrusions compressing the capillary lumen [70].

The glymphatic system (GS) is a fluid exchange and drainage system supported by glial cells [71]. The GS includes the entire peripheral vascular system and consists of periarterial cerebrospinal fluid inlets and perivenous interstitial fluid (ISF) outlets [71]. Astrocytic end plates mediate the transport of metabolites via aquaporin-4 (AQP4) channels [72]. This transport system was confirmed with the use of fluorescent tracers administered into the brain parenchyma, which showed deposition in the meningeal lymphatic vessels and ultimately the deep cervical lymph nodes (dCLNs) [73]. Overall, the glymphatic system facilitates transport and clearance of metabolic waste under normal physiologic conditions [74], while during SAH, the glymphatic system is involved in the clearance of neurotoxic solutes, pro-inflammatory cytokines, and erythrocytes [71,75].

After the initial SAH event, blood and its metabolites rapidly leak into the PVS. Blood in the PVS diffuses into the perivascular parenchyma via the GS, leading to a cascade of glial activation, neurotoxicity, and widespread microcirculatory dysfunction in the brain [76]. A recent study performed by Chen et al. found that meningeal lymphatics drained extravasated erythrocytes into the cerebral spinal fluid [75]. Following experimental ablation of meningeal lymphatics via the injection of Visudyne, there was a measurable decline in RBCs found in the cervical nymph nodes [75]. Additionally, following meningeal ablation, neuroinflammation and neurologic deficits were increased in a rodent model. Persistent malfunction of glymphatic and meningeal drainage was observed in an SAH mouse model by Pu et al. [77]. Decreased tracer was observed in the SAH mice compared to controls in meningeal lymphatic vessels and dcLNs [77], suggesting that SAH impairs the ability of meningeal lymphatic vessels to drain cellular debris, immune cells, and inflammatory mediators [78,79]. Additionally, glial cells lining the glymphatic regions become activated in SAH mice, as determined by increased levels of IL-1β, IL-6, and TNF-α expression [77]. This process results in AQP4 upregulation surrounding the arteries, while the AQP4 expression remains the same at the drainage sites around the veins [80]. Consequently, impaired PVS and ISF flow reduces metabolite and hemorrhagic clearance, leading to intermediate injury via immune cell accumulation [81], BBB dysfunction [82], neuronal apoptosis [77], vasculitis [76], cerebral edema [83], and acute hydrocephalus [84].

In addition to the glymphatic system, there is another drainage system called the intramural periarterial drainage (IPAD), which has been described as functioning in parallel with the glymphatic system. This system can also be affected during subarachnoid hemorrhage (SAH). Interstitial fluid (ISF), which is the fluid found between brain cells, can drain from the brain along the basement membranes of arteries through the IPAD pathway. Disruptions in the IPAD pathway can lead to problems with proteostasis in the brain, such as in cerebral amyloid angiopathy or Alzheimer’s disease. A study by Sun et al. [85] examined disturbances in the IPAD pathway after SAH in rats. In that study, the authors injected dyes of different sizes into the cisterna magna, a fluid-filled compartment at the back of the brain in the posterior cranial fossa. The cisterna magna is one of the subarachnoid spaces, which is filled with cerebrospinal fluid (CSF) and surrounds the brain and spinal cord. Sun’s group observed that these dyes entered the brain through periarterial channels and were cleared through the basement membranes of the brain’s associated capillaries. Interestingly, different molecular weight tracers showed different patterns of clearance. SAH significantly disrupted the IPAD pathway, causing enlargement of spaces around the blood vessels where ISF clearance was reduced. This effect was related to endothelial cell death, activation of astrocytes (a type of brain cell), increased levels of matrix metalloproteinase-9, and loss of type IV collagen in the basement membrane. Consequently, experimental SAH in rats has been found to significantly disrupt the IPAD pathway, which could have important clinical implications for SAH.

A hallmark of SAH is BBB degradation [86] in the form of endothelial and pericyte dysfunction [87]. Extracellular matrix proteins are also degraded following SAH-mediated degradation of endothelial cells [88].

Cytokines are locally released following SAH-mediated neuroinflammation to degrade cell adhesion molecules (i.e., ICAM-VCAM). Moreover, SAH-mediated neuroinflammation can cause a leaky BBB, which has been shown to decrease tight junction proteins such as ZO-1, occludin, and JAMa.

Pericytes are also important regulators in response to the change in cerebral blood flow with respect to local neuronal activity [89,90,91]. Pericytes also regulate and maintain the BBB by supporting tight junctional proteins [92]. Three to five days after brain injury, glycocalyx breakdown and endothelial dysfunction lead to increased BBB permeability [93]. BBB leakage allows for plasma protein extravasation, such as albumin, activating astrocytes, which in turn disrupt the neurovascular coupling [94]. More importantly, due to increased BBB leakage, extravasation of plasma proteins into the brain parenchyma is a major contributor to brain edema [94,95].

Pericytes amplify the inflammatory response by increasing their expression of microglial markers [96], upregulating periarterial AQP4 anchored in astrocytic end feet [97,98] and enabling extravasation of immune cells [99]. Smooth muscle cells (SMCs) undergo phenotypic transformation and migrate to the subepithelial layer [100]. IL-1β stimulates SMC proliferation in cerebral arteries and arterioles [101] and pericyte invasion of capillary networks, producing basement membrane remodeling [102]. Migration of transformed pericytes and SMCs increases the propensity for vessel spasm [103,104,105]. EC apoptosis starts around 24 h following SAH [106].

Astrocytes also modulate local vasoconstriction and vasodilation by releasing vasoactive compounds that stimulate 20-hydroxyeicosa-tetraenoic acid (20-HETE) production in pericytes [107], Intracellular Ca²⁺ concentrations in pericytes are also regulated by astrocytes promoting contraction [89,108]. Upregulation of AQP4 in astrocytes accelerates the formation of cytotoxic brain edema, leading to intracellular water accumulation [109,110]. Early in SAH, cellular edema in astrocytes produces an influx of ionic and vasogenic edema, leading to parenchymal edema [95]. Ionic edema develops when osmotic forces in the vasculature propel plasma through the vessel wall into the CNS [95]. The trans-endothelial Na⁺ gradient increases across the endothelium, leading to Na⁺ accumulation in the brain parenchyma [111]. This effect is magnified with early-onset CSF hypersecretion by the choroid plexi during SAH [112,113] and declining ISF efflux [114]. The large increase in parenchymal influx from extravasation and hydrocephalus exceeds the clearance rate provided by the glymphatic system. Tight junction breakdown and EC dysfunction contribute to vasogenic edema (e.g., extravasation of plasma proteins) [95]. Increased BBB permeability, cell death, and microvascular spasm and decreased ISF waste clearance escalate shifts in ion and water balance [95,115,116]. This process leads to neurovascular uncoupling, resulting in increased neuron susceptibility to terminal injury from cortical spreading depolarizations [117], excitotoxicity [118], neuronal apoptosis [81], and secondary brain injury [119].

The neurovascular unit (NVU) is comprised of the BBB (e.g., ECs, pericytes, SMCs, astrocytes) and its communication with neurons and microglia [120]. The NVU serves to modulate pressure to local regions under normal physiologic conditions, such as regions that process reading, arithmetic, languages, etc. The NVU can also respond to pathophysiologic stimuli, such as seizures or CSD. The neurovascular unit is coupled when the local blood supply matches neuronal demand via modulation of vascular diameter [121]. The cellular interactions in the NVU dynamically regulate this activity [122]. Glutamate released from neurons stimulates nearby astrocytes and pericytes, generating vasoactive molecules [123]. The concentration of vasoconstrictor and vasodilator mediators determines the tone of the surrounding vasculature, modulating CBF [123]. In this model, neurons are “pacemakers” within the NVU model in that they regulate CBF [122]. For example, administration of catecholamines (e.g., dopamine, norepinephrine, and epinephrine) to neurons modifies EC tight junction protein production, thus changing BBB permeability [124]. All components in the NVU may modulate BBB maintenance [125]. Similarly, these components all coordinate to tightly control the CNS ionic microenvironment and ensure optimal neuronal functioning [126]. These processes include having specialized functions for neurotransmitters, maintaining low protein concentrations, preventing CNS exposure to neurotoxins, and limiting inflammatory processes [126]. The main function of astrocytes in the NVU is to regulate nutrient exchange by changing the tight junction density [127,128,129].

BBB breakdown following SAH disrupts the NVU, leading to neuronal hyperexcitability [130]. Neuroinflammation occurring in the CNS decreases the seizure threshold due to rapid changes in glutamate and γ-aminobutyric acid (GABA) receptor phosphorylation and channelopathies [131,132]. In rodent and pig models, BBB breakdown synchronized neuronal activity and increased seizure activity [133,134]. Albumin extravasation contributes to neuronal excitotoxicity by disrupting astrocytic K⁺ buffering capacity [130]. Albumin injection in naïve rats downregulated astrocytic Kir4.1 channels, leading to transient spiking activity [135]. These findings support neuroinflammation and albumin influx as drivers of neuronal network hyperexcitability [127].

During the intermediate phase (3–5 days post-SAH) neurovascular coupling is compromised following BBB dysfunction [136]. The brain tissue is particularly vulnerable during this period (3–5 days post-hemorrhage); cerebral vasospasm (CVS) is likely to occur during this period and further exacerbate damage to the brain parenchyma [137]. Neurovascular uncoupling occurs when there is a mismatch between blood supply and neuronal demand [38]. Uncoupling is exacerbated by neuronal hyperexcitability (e.g., increased metabolic activity), dysregulated vascular contractility, and decreased metabolic waste clearance, thus reducing the neuronal energy supply, leading to apoptosis [138]. The glymphatic system continues to deteriorate as SAH progresses, with loss of gap junctions (GJ) connecting neighboring astrocytes [139,140]. GJ loss limits neurotransmitter uptake, ion buffering, and glucose distribution for stable neuronal activity [141,142,143].

Loss of astrocytic GJs contributes to astroglial network compromise and subsequent astrocytic apoptosis [144,145]. However, loss of the astrocytic GJ loss diminishes the spread of Ca²⁺ waves, preventing synchronized activity between neurons and vascular SMCs [146]. Concurrently, disconnection between neurons and the supporting astroglial network hinders the neuronal energy supply [147]. In the setting of hypoglycemia or periods of neuronal hyperactivity, astrocytes monopolize the energy supply [148,149]. This cellular strategy provides another energy source (e.g., astrocyte–neuron lactate shuttle) for neurons during periods of excessive energy demand [150]. In SAH, astrocytic apoptosis is favored to precede neuronal apoptosis [145].

The deregulated neuronal activity of astrocytes depletes the neuronal energy supply and disrupts Na⁺–K⁺ pumps, which maintain ion homeostasis [151]. The means for neuronal ATP production are also impaired without the supporting astroglial network [151]. Injured neurons incur further injury from reactive Ca²⁺ influx, leading to large amounts of glutamate release, triggering local depolarizations [152]. Cortical spreading depolarizations (CSDs) consist of recurrent waves of neuronal and glial depolarizations that propagate widely from the onset zone [153]. CSDs begin under hypoxic conditions, strained energy supplies (e.g., glucose), and exposure to oxyhemoglobin following SAH [154,155]. CSDs peak at days 5–7 following aneurysmal SAH [156]. Changes in the neuronal microenvironment lead to massive glutamate release, loss of membrane potential, and CSD throughout the brain parenchyma [157]. If injured neurons cannot restore membrane potentials using Na–K⁺ pumps, the associated neurons and astrocytes swell and distort the dendritic connections [158]. CSDs eventually evolve into epileptiform discharges or pathologic changes in electrical potentials, silencing brain electrical activity [159]. Epileptiform discharges transmit greater disturbances in ion homeostasis between the intracellular and extracellular environments, resulting in neuronal swelling and glutamate receptor upregulation [159,160]. Underlying epileptiform discharges are neurons stuck in field potentials that are less than the inactivation threshold for channels generating action potentials [154]. Thus, neurons fire at high frequencies, producing moderately sustained depolarizations and less hyperpolarization [154,161].

When metabolic demand exceeds the energy supply provided by the neurovascular unit (e.g., glucose delivery, cerebral blood flow), neuronal ischemia and apoptosis occur [162]. Within minutes, neuronal necrosis surpasses apoptosis as the primary form of cell death within the brain’s parenchyma [163]. The neuroinflammatory response is caused by an increase in neuroinflammatory signaling agents, such as IL-6, PAF, T-cells, and macrophages. The cytokines/chemokines involved are p53, Fas/FasL, tumor necrosis factor receptor, caspase 9 and apoptosis inducing factor [164]. These cytokines and chemokines lead to increased cell death of the brain’s parenchyma [165].

During the late or delayed stage (5–14 days post-SAH), CSDs peak in their occurrence. These peaks are associated with loss of neurovascular autoregulation. This loss is due to deranged NO/NOS signaling following changes to eNOS, nNOS, and iNOS [166]. During this phase, RBCs begin to break down/lyse within the subarachnoid spaces and increase oxyhemoglobin and ferritin concentrations within the brain parenchyma, causing further cerebral vasospasm [167].

The mass effect within the cranial vault physically displaces the brain parenchyma. The displaced parenchyma moves from a higher-pressure region (adjacent to the growing hematoma) to lower-pressure/resistance region(s) (e.g., lumens [openings] within the cranial vault). This process is problematic since, within the cranial vault, these openings are occupied by critical structures (e.g., brainstem, cranial nerves, vertebral arteries and veins) [168]. The brainstem controls functions such as respiration, blood pressure regulation, and other autonomic functions required for life. Compression of the brainstem leads to the loss of these critical autonomic functions [169,170,171,172].

Initially described by Ecker and Riemenschneider in 1951 [173], the association of CVS following SAH has been well documented. However, the exact mechanisms of CVS following SAH have been contested within the recent literature [174]. Previously, the accepted model of CVS, as described by Allcock and Drake [175], was that it occurred in the setting of focal ischemia experienced by the brain parenchyma during SAH and with a peak occurrence at 5–14 days post-initial hemorrhagic event [176]. Additionally, there exists a positive correlation between hemorrhage volume (HV) and the severity of CVS; this correlation has been recognized for its clinical utility and is used as the basis for the Fisher scale [177]. Recently, an ongoing clinical trial examined deferoxamine (i-Def), an iron chelating agent that sequesters the iron component of red blood cells. When i-Def was introduced into the brain’s parenchyma during a hemorrhagic event, initial results from second-phase clinical trials [178] were promising for reducing stroke sequalae [179]. If HV was less than 10 mL, the iatrogenic complications can outweigh any benefit that otherwise would be conferred by i-Def [180].

The link between cerebral vasospasm following the accumulation of blood degradation byproducts has been well established for several decades and was described by Toda and Ohta [181]. Recently, it has been shown that a potent inflammatory marker, oxyhemoglobin, is implicated in CVS, replacing the previously targeted methemoglobin and bilirubin as inflammatory markers [182,183]. The exact mechanism by which these substances induce CVS is still not well understood, although several features have been described. When injected intrathecally, oxyhemoglobin has been shown to induce CVS [174]. The mechanisms by which this outcome occurs is through the modification of normal expression of eicosanoids; oxyhemoglobin will increase the production of PGE₂ and decrease PGI₂ levels. Both eicosanoids are important in maintaining vascular tonicity. When oxyhemoglobin contacts methemoglobin, it spontaneously releases superoxide, which in turn causes lipid peroxidation, as well as vasoconstriction [174].

Additional substances released into the brain parenchyma during structural compromise of the vessel wall come from the spillage of intervascular wall components (intracellular enzymes and proteins) [189] that are cytotoxic to surrounding CNS tissue. Within minutes, there are gross anatomical and cellular changes to the CNS vasculature, e.g., mechanical/physical compression of neighboring/adjacent vessels, occluding their internal lumen(s), as well as compression of CNS tissue [190,191]. Regarding the cellular changes, notable is the disruption to chemosignaling agents (vasodilators/vasoconstrictors). These signaling agents under normal physiologic conditions are kept in a delicate balance to maintain the CNS vessels’ ability to autoregulate their tone [192].

Following the loss of cerebrovascular autoregulation, ICP equalizes to diastolic blood pressure (DBP) [191]. Given that DBP is several fold greater than normal CNS pressures (80–100 mm Hg and 7–15 mm Hg, respectively), the pressure differential can cause lateral displacement and subsequent compression of CNS tissue [193].

Ultimately, the compounding anatomical and cellular changes further contribute to a loss of micronutrients (perfusion) to local tissues and lack of clearance of toxic metabolic byproducts [191,194]. Within minutes of changes to perfusion, the cell’s ionic transmembrane gradient, which is crucial for normal neuro-signaling, becomes deranged [195]. This outcome follows the failure of adenosine triphosphatase’s ability to maintain the gradient in hypoxic conditions and thus its reliance on relatively ineffective anerobic pathways.

Following SAH, arterial vasospasm can occur, independently impairing perfusion (excluding non-spasmodic causes, e.g., clots, rupture of an atherosclerotic plaque, or embolus). Traditionally, this outcome has been detected by means of transcranial ultrasound (TCD) performed at the patient’s bedside approximately 2–3 days post-initial insult [196,197]. TCD is performed when vasospasm is suspected [198]. Currently, clinicians rely upon alterations in mentation or focal neurologic changes to escalate to imaging modalities for affected patients. In practice, these include shifts in the patient’s level of consciousness (neurobehavioral changes) or any new-onset focal neurologic defects [199]. However, this process can be challenging in patients who are intubated and/or comatose, especially for junior clinicians [200].