Neurogenic inflammation after traumatic brain injury and its potentiation of classical inflammation

Extensive research has shown that levels of SP rise acutely following TBI in both pre-clinical animal models and in human tissue. Virtually, all blood vessels of the body are surrounded by sensory nerve fibers that contain SP [84]. Cerebral arteries, in particular, appear to receive a dense supply of these nerve fibers, and our studies in TBI have demonstrated that perivascular SP immunoreactivity increases in pre-clinical models, irrespective of injury model [13, 85, 86], and also in humans [87]. It appears that SP is released early following TBI, with increases noted in the plasma at 30 min following TBI in rodents [13]. Furthermore, this release of SP appears to depend on the magnitude of the insult, with a graded increase in SP immunoreactivity seen with increasing severity of injury [85, 88]. Indeed, SP appears to be a key injury marker, as levels in the plasma over the first 4 h following injury are significantly correlated with early mortality in clinical populations, with non-surviving TBI patients showing significantly higher levels than survivors [88].

Moreover, it has been shown that attenuating SP activity following TBI is beneficial to outcome [89]. The first demonstration of neurogenic inflammation in TBI showed that depletion of sensory neuropeptides by pre-treatment with capsaicin results in the attenuation of post-traumatic BBB permeability, edema formation, and improved functional outcome [11]. Later studies, specifically targeted SP by administering an NK1 antagonist showed beneficial effects in both male [13] and female rats [90], with a significant attenuation of post-traumatic BBB permeability and a resultant significant reduction in edema formation with improvement in motor and cognitive outcome.

It appears likely that the initial release of SP from sensory neurons following TBI may be mediated by mechanical activation of members of the transient receptor potential (TRP) family, predominantly TRPV1 and TRPA1 [91]. Like all TRP receptors, TRPV1 and TRPA1 are comprised of six-transmembrane proteins that assemble as tetramers to form cation-permeable pores [92, 93]. Their activation allows the influx of cations, primarily sodium and calcium, triggering the release of neuropeptides [94, 95]. TRPV1 appears to be co-expressed in most if not all of TRPA1 expressing dorsal root ganglion neurons [96, 97], with co-expression of both these receptors with neuropeptides including SP [98]. Indeed, suppression of the TRPV1 receptor, with the antagonist capsazepine, has been shown to significantly reduce SP levels in a number of inflammatory models, including a model of sepsis [99], alcohol-induced gastric injury [100], and formalin-induced asthma [101], with the latter study showing a similar reduction in SP with administration of the TRPA1 antagonist, HC-030031 [101]. Furthermore, activation of both TRPV1 and TRPA1 [102‐104] has been linked to increased vascular permeability, a key downstream effect of SP release. TRPV1 immunoreactivity is prominent in astrocytes and pericytes, which are closely associated with the vasculature, as well as neurons [105], with the administration of capsazepine able to reduce BBB disruption following an ischemia-reperfusion injury [106].

TRPV1 and TRPA1 channels are considered as polymodal receptors that are activated by a wide range of stimuli. For TRPV1, this includes capsaicin (the active ingredient in chilies) [107], heat (43–52 °C) [108], protons [107], bradykinin [109], prostaglandins [110], and arachidonic acid metabolites amongst others [111]. For TRPA1, agonists include exogenous noxious agents such as components of wasabi and cinnamon [98], oxidized lipids [112], protons [113], and potentially cold (<17 °C) [113, 114]. Notably, both TRPV1 and TRPA1 are also putative mechanoreceptors. Early reports identified that the long ankyrin repeat region within the N-terminal domain of TRPA1, which assists in anchoring the receptor to the plasma membrane, could form a spring-like structure to sense mechanical forces [115, 116]. Indeed, TRPA1 knockout mice were found to be less sensitive to low-intensity mechanical stimuli compared to wild-type mice, and responses to high-intensity mechanical stimulation were notably impaired [113]. Furthermore, inhibition of TRPA1 via either gene knockout or treatment with the antagonist HC-03001 reduced mechanically induced action firing in dermal C fibers [117, 118], wide dynamic range, and nociceptive-specific neurons within the spinal cord [119] and mechanosensitive visceral afferents in the colon [120, 121]. Studies have also suggested that TRPV1 detects mechanical stimuli in a variety of tissues including the urothelium cells of the bladder [122], colonic primary afferent neurons [123], renal pelvis [124], and retinal ganglion cells [125, 126]. The threshold for the stimulation of both TRPV1 and TRPA1 appears to be lowered when there is pre-existing inflammation. Indeed, blockade of TRPA1 was only effective in reducing low-intensity mechanical stimulation of spinal neurons in animals with inflammatory arthritis [119], and application of a TRPV1 antagonist reduced Aδ-fiber unit responses only in inflamed skin [127].

In regard to TBI, a report by Zacest et al. showed that TBI mechanical stimulation of sensory nerves, presumably via TRPV1 and TRPA1, facilitates the perivascular release of SP, with SP co-localizing with amyloid precursor protein positive (injured) neurons that supply the blood vessels [87]. Apart from the initial external mechanical insult to the brain, another acute mechanical stimulus could be related to a brief but highly significant spike in blood pressure (BP) seen following injury. In a sheep model of TBI, BP was reported to rise markedly immediately after impact, peaking at a MAP of 176 mmHg before gradually returning to baseline over a 10-min period [128]. Similar findings have been shown in other models of experimental TBI [129‐131]. TRPV1 receptors have indeed been shown to respond to increased intraluminal pressure [132, 133], with Scotland et al. showing that the normal myogenic response to elevation of increased transmural pressure in mesenteric small arteries in vitro was suppressed with the application of the TRPV1 antagonist capsazepine. As such release of neuropeptides including SP may be one of the earliest responses to TBI, highlighting its integral role in facilitating the inflammatory response.

SP induces and augments many aspects of the classical inflammatory response (Fig 1), including leukocyte activation, endothelial cell adhesion molecule expression, and the production of inflammatory mediators such as histamine, nitric oxide, cytokines (such as IL-6), and kinins [14, 15]. SP is a potent mast cell activator [134]. Mast cells are found within the brain on the adluminal side of the BBB and in the leptomeninges [135], with their degranulation found to potentiate excitotoxicity [136] and augment and prolong numerous vasoactive, neuroactive, and immunoactive cellular and molecular responses to injury [137, 138].

SP also directly activates microglia and astrocytes. Injury induces the expression of NK1 receptors on astrocytes, and their activation is thought to contribute to the transformation to reactive astrocytes, with the resultant production of inflammatory mediators such as cytokines, prostaglandins, and thromboxane derivatives [139‐141]. Similarly SP can promote microglial activation, initiating signaling via the NFκB pathway, which leads to the production of pro-inflammatory cytokines [142], with microglia producing IL-1 in response to SP [143]. Moreover, following TBI, NK1 antagonists have been shown to significantly reduce the production of the pro-inflammatory cytokine IL-6, as well as to decrease microglial proliferation [144].

Apart from directly augmenting the local classical inflammatory response, SP-related BBB disruption may also play a role in perpetuating the inflammatory response. The BBB is a highly selective barrier formed by a layer of endothelial cells joined together by tight junctions including proteins such as claudins, occludin, junctional adhesion molecules (JAMs), and zonula occluden (ZO) proteins, which are supported by the end-feet of astrocytes that act to support and enhance the tight junctions [145]. Tight junctions are present at the apical end of the interendothelial junction and closely rely on the integrity of the corresponding cadherin junction located in the basolateral region. Ultrastructural studies of cerebral endothelial cells indicate the circumferential arrangement of the TJs which provide the barrier formation required to actively restrict the movement of small hydrophilic molecules such as sodium and particular permeability tracers between cells [146]. Given the restrictions on paracellular transport, cerebral endothelial cells have tightly controlled mechanisms that enable the bidirectional transcellular passage of essential molecules and the efflux of potentially neurotoxic substances and waste products [147]. Smaller molecules can be ferried across via carrier-mediated transport or ion pumps while macromolecules employ transcytosis, which can be either receptor-mediated transport (RMT) or adsorptive-mediated transport (AMT), where positively charged macromolecules are nonspecifically transported across the cerebral endothelium [148]. Although the cerebral endothelium has relatively few pinocytotic vesicles compared to peripheral endothelial cells [149], they can still transport macromolecules via one of three vesicles: clathrin-coated pits, the most numerous and through which most of the RMT occurs, the smaller and less numerous caveolae, capable of both AMT and RMT, and large macropinocytic vesciles with nonspecific cargo [150].

Following TBI, the BBB is disrupted and this encompasses not just potential disruption to tight junctions but also increased transcytosis and altered transport properties [151‐153]. This increase in the transcellular permeability of the BBB permits the extravasation of proteins and solutes from the cerebral vasculature into the extracellular space within the brain, promoting edema formation, perpetuating the inflammatory response, and causing further neuronal injury. Importantly, it appears that this transcytotic activity is the initial alteration to the BBB following TBI, which is then followed by later loss of tight junctions and movement via the paracellular pathway [151, 152]. An early study by Povlishock (1978) found an increase in BBB permeability as early as 3 min following a mild central fluid percussion injury, despite the apparent integrity of the endothelial tight junctions [154]. Notably, endothelial vesicles assume a modified appearance and initiate the formation of extensive networks for intracellular transport after injury, particularly in the setting of inflammation within the CNS [155]. Indeed, ultrastructural studies have shown that within minutes of brain injury, endothelial caveolae are significantly increased in vessel segments showing loss of BBB integrity [152, 155], with evidence for increased vesicular formation in the BBB of TBI patients [156, 157]. In a rat cortical cold injury model that produces a pure vasogenic edema, an increase in expression of caveolin-1, the key protein found in caveolae, occurred prior to the loss of tight junctions, represented by a reduction in the expression of occludin and claudin-5 [152]. Notably, caveolin-1 expression was seen in blood vessels which exhibited increased permeability to proteins, indicating that this may be the initial mechanism by which the BBB is disrupted [152]. These findings were replicated in a controlled cortical impact model of TBI, where caveolin-1 was increased at 1 day following injury, prior to a decrease in claudin-5 which occurred at day 3 following injury [151].

SP is known to promote increased permeability of the BBB leading to increased extravasation of vascular protein into the brain extracellular space. Indeed, injured animals show a strong co-localization of SP immunofluorescence with a marker of vascular permeability, Evan’s blue, within the cortical vasculature at 5 h post-TBI [13]. Administration of an NK1 antagonist also leads to a dose-dependent decrease in permeability of the BBB following TBI, with an accompanying reduction in cerebral edema [13]. The exact mechanisms by which SP promotes an increase in BBB permeability are less clear. There are previous reports that application of SP can decrease the expression of ZO-1 and claudin-5 in cerebral capillary endothelial cells [158], but immediately following TBI tight junctions are intact. Alternatively, SP may initially act to increase transcytosis via activation of transport via caveolae.

Caveolae have been shown to mediate the transport of molecules such as albumin, transferrin, insulin, low-density lipoproteins, cytokines, and chemokines through the specific localization of corresponding receptors within the caveolae coats [159, 160]. Indeed, caveolin-1 knockout mice were unable to transcytose albumin as evaluated via gold-labeled immunostaining [161, 162], supporting a key role for caveolae in the early BBB changes seen following TBI, and consistent with the increases seen in expression of caveolin-1 in the immediate phase following injury [151‐153]. Caveolae constitute an entire membrane system responsible for the formation of unique endo- and exocytotic compartments [163]. During transcytosis, caveolae “pinch off” from the plasma membrane to form vesicular carriers that rapidly and efficiently shuttle to the opposite membrane of the endothelial cells, fuse and release their contents via exocytosis [164]. In addition to the structural caveolin proteins, a number of signaling molecules, including specific receptors, are known to localize to the caveolae pit [160, 165]. Receptor tyrosine kinase, G-protein-coupled receptors, transforming growth factor-beta (TGF-β) type 1 and 2, certain steroid receptors and enzymes have a confirmed presence within endothelial caveolae and may play a role in facilitating macromolecular transcytosis [166‐168]. Of particular note, the NK1 receptor, to which SP preferentially binds, has been reported to localize within the endothelial caveolae and can be manipulated to upregulate or relocate upon stimulation, indicating that its presence and function is dynamic and subject to the environment [169, 170]. Indeed, stimulation of the NK1 receptor within caveolae by SP causes PKCα to relocate to caveolae [170], with this process previously shown to regulate the internalization of caveolae [171], the first step in transcytosis. Although not directly studied for the NK1 receptor, in vitro studies have demonstrated increased receptor-mediated transendothelial transport of target molecules in the setting of inflammation [172]. This suggests that the activation of a caveolae-housed receptor may also internalize additional proteins in their cargo, such as albumin, facilitating their entry into the brain.

Within the CNS, albumin is able to activate microglia and astrocytes leading to the release of pro-inflammatory cytokines and chemokines (e.g., IL-1β, TNFα, MCP-1, CXC3L1), glutamate, and free radicals promoting neuronal injury and amplifying the classical inflammatory response [173‐178]. Although the majority of these studies apply albumin to isolated glial cultures [174‐177], Chacheaux et al. were able to demonstrate that direct application of a solution containing bovine serum albumin to the rat cortex produced similar changes in gene expression when compared to application of an agent (deoxcycholic acid) that directly opens the BBB [179]. Microarray analysis showed a comparable gene expression profile to either treatment, with an early and persistent upregulation of genes associated with immune response activation, including NF-κB pathway-related genes, cytokines, and chemokines (IL-6, CCL2, CCL7) [179]. Indeed, recent evidence suggests that following activation by albumin, microglia are neurotoxic, with media taken from microglial cultures exposed to albumin promoting the upregulation of caspase 3 and 7 in cerebellar granule neuronal cultures with an accompanying increase in cell death [174]. Furthermore, when exposure to albumin was combined with TLR4 activation via lipopolysaccharide (LPS), a synergistic increase in the release of pro-inflammatory cytokines was noted from cultured microglia [174]. As the neuronal injury associated with TBI leads to the production of DAMPs [180] that act as TLR4 agonists, influx of albumin through a disrupted BBB would lead to more potent activation of microglia. This provides another key intersection between the classical inflammatory response, in TLR4 activation by DAMPs, and the neurogenic inflammatory response, with SP release promoting albumin influx via caveolae promoting further activation of resident immune cells.

The effects of SP on the BBB relates not only to increases in permeability but also on promoting immune cell trafficking into the CNS. Activation of SP’s preferred receptor, NK1, has been shown to enhance leukocyte migration by exerting a chemotactic effect on monocytes and neutrophils [181‐183], increasing the expression of adhesion molecules on endothelial cells [184‐187] and augmenting local chemokine production [188]. Indeed, application of SP to cerebral endothelial cultures led to a dose-dependent increase in ICAM-1 expression [184], with this associated with an increase in T lymphocyte adherence, suggesting facilitation of immune cell movement across the BBB [187]. Thus, the release of SP following TBI may facilitate the influx of peripheral immune cells like T cells, macrophages, and neutrophils into the CNS, which then further augments the local neuroinflammatory response via production of pro-inflammatory cytokines, reactive oxygen species, and metalloproteinases.