4.1 Excess iron is regulated by three distinct pathways.
4.1.1 Hemosiderin and transferrin binding.
Accumulation of red blood cells (RBCs) has been observe in fluid percussion injury (FPI) . These RBCs come from blood vessels ruptured by the transient mechanical forces produced by the impact, in which hemorrhage lasted for several days resulting in a substantial aggregation of RBCs especially in the epidural, subarachnoid, and ventricular spaces . Failure to clear these RBC aggregates may cause the formation of an impacted blood mass and can result in an enduring mass effect. With time RBC aggregates can penetrate neighboring tissue as we observed in our findings (Fig. 1). This may be due to their low resistance and viscoelastic properties as previously noted . Interestingly, the most penetration of RBCs was observed at subarachnoid spaces as opposed to any other area. Since the subarachnoid space facilitates fluid flow into the brain, RBCs may be using this current to enter brain tissue . These RBCs homing in tissue cannot survive for long time, they eventually undergo hemolysis to release free irons. We found that iron remains ferrous for only a few hours following injury as ferric iron was observed as early as 1-day post-FPI (Fig. 4). This explanation is in agreement with the findings of others . Free iron, whether as a ferrous cation or an unbound ferric species, is readily absorbed by cells and can be very toxic . The unpaired electrons make free iron highly chemically reactive and, through the Fenton-Haber-Weiss reaction, catalyze the formation of free radicals (Fig. 8). This may be a reason for iron’s rapid transformation from ferrous to ferric, and finally to the bound form by the body.
Although transferrin increased following injury (Fig. 7.D), generally, transferrin becomes saturated when ~70% is bound with iron . Western blot analysis showed greater F-LC levels in plasma than transferrin (Fig. 7.D). When this main iron binding protein in the blood is so far extended that it cannot bind any more iron, excess catalytic iron must be stored by ferritin or transformed ferritin, i.e., hemosiderin, to prevent iron toxicity. Significant amounts of hemosiderin collections, ranging in size, were found at all bleed sites (Fig. 7.B). These results reveal hemosiderin binding as a pathway in iron management specially reserved for superfluous excesses of iron. Iron as hemosiderin is not readily available for release thereby making this binding very stable. Like ferritin binding, iron release from hemosiderin may possibly be achieved by lysosomes (Fig. 8). Extracellular hemosiderin complexes may also follow suit and be degraded by strong digestive enzymes. More research is needed on the fate of iron following hemosiderin binding and on understanding the circumstances and elements involved in its release; it is likely lysosomes are involved.
4.1.2 Sequestration by iron regulatory proteins.
Russell et. al. (2019) described a time-dependent induction of the iron regulatory proteins LCN2, HO-1, and F-LC following an FPI-induced traumatic brain injury . We have observed similar results in rats that have undergone FPI alone but a time shift in expression profiles following prior alcohol consumption. In both FPI and EtOH + FPI situations we have observed a similar expression pattern for LCN2 (Fig. 5). By immediately trafficking and sequestering iron, LCN2 expression stimulates anticipation for possible incoming or excess iron to prevent its congestion. This result demonstrates that LCN2 induction may not depend on the type of injury but rather by the inflammation associated with the injury. Therefore, if the injury causes any release of inflammatory stimuli, LCN2 becomes expressed. This result demonstrates that combined, alcohol and FPI injuries do not necessarily exacerbate the degree of inflammation, but rather prolong its persistence. However, failure to resolve this inflammation can lead to a chronic inflammatory state in the CNS and manifest debilitations in attention and cognition . Therefore, by maintaining LCN2 expression, alcohol consumption may transform FPI-induced inflammatory properties. In this way, LCN2 may act as a master switch for the induction of subsequent iron regulatory proteins. LCN2 may occupy a greater role in iron regulation than has been so far suggested.
One important regulator of HO-1 expression is the nuclear transcription factor Nrf2 . Nrf2 also controls production of the antioxidant proteins that protect against the oxidative damage caused by an insult. Therefore, HO-1 is directly related to the presence and extent of oxidative damage. We have previously shown that inflammation, from both FPI and the resulting hemorrhage, can trigger oxidative damage . Since LCN2 expression is determined by inflammation and HO-1 is mediated by oxidative stress, HO-1 induction is expected to occur after LCN2 under FPI (Fig. 5). However, alcohol presence causes HO-1 to express concurrent with LCN2 (Fig. 5). EtOH + FPI must exacerbate oxidative damage enough to cause this shift to earlier protein expression.
Interestingly, F-LC was observed as clusters. F-LC clustering may be the result of a partial unfolding of the protein shell in preparation for iron binding . Unlike ferritin heavy chain (F-HC), F-LC has no ferroxidase activity, instead it is involved in the transfer of electrons across its protein cage which allows F-LC staining to mark unfolded shells . Notably, we observed a delayed ferritin response following FPI, peaking at 7-days (Fig. 5). These results demonstrate that ferritin is the final regulatory protein involved in iron management, sequestering any remaining unbound iron for accessible storage. Even hemosiderin, observed in 1-day post-FPI, precedes significant ferritin expression (Fig. 4). Furthermore, this delay shows ferritin to be induced primarily by iron release and secondarily by heme presence. Low ferritin levels under EtOH alone suggest that ferritin is nominally induced by stress. However, EtOH + FPI caused early, increased, and sustained ferritin expression. Due to the excess RBC accumulation following these combined injuries, much of this ferritin may be being excreted and transformed to hemosiderin for more stable storage. These increased ferritin levels also imply high iron concentrations in the CNS.
4.1.3 IRPs also function in the extracellular space.
Interestingly, LCN2, HO-1, and F-LC were also expressed directly atop bleed sites, interacting with RBCs (Fig. 4). This presence may be explained by the mechanical forces caused by the impact bursting cells and releasing intracellular proteins into the extracellular space. As demonstrated, alcohol may also help facilitate secretion of these proteins by increasing their basal levels. There is evidence of LCN2, HO-1, and F-LC secretion in other organ systems. Hepatic cells have been shown to secrete ferritin, while HO-1 has been found in various extracellular, fluid filled compartments and LCN2, as a mediator of inflammation, is routinely released by various cell types, most notably immune cells [44-46]. Extrapolating these findings and relating then to the CNS space, HO-1 may be in the CSF while LCN2 gets secreted by astrocytes and microglia, and ferritin released by neurons. Then protein targeting mechanisms may explain their localization at sites of RBC aggregation. More research on the default expression levels and locations of LCN2, HO-1, and F-LC in the CNS as well as their intrinsic signaling sequences help explain this phenomenon.
LCN2/HO-1 colocalization can indicate that even HO-1 may not be constrained to expression following FPI, instead it can be induced by the oxidative stress accompanying inflammation . Meanwhile, LCN2/F-LC colocalization suggests LCN2 expression may recruit F-LC generation in preparation for iron sequestration. The lack of microglial expression of these iron regulatory proteins suggests microglia may not be involved in the lipocalin 2/heme oxygenase 1/ferritin system of iron management. Another limitation of this study was confining it to 7-days. However, previous studies have shown that beyond 7-days, LCN2, HO-1, and F-LC levels comparatively return, or being to return, to basal levels in most cells of the CNS . A notable exception are microglia which have shown elevated HO-1 expression as late as 30-days relative to controls under a moderate cortical impact . In this effect, we argue that microglial involvement may create a separate pathway for iron management, removed from that created by the iron regulatory proteins of the remaining CNS cells.
4.1.4 Microglial phagocytosis.
To our knowledge, this is the first demonstration of RBC phagocytosis by microglia and subsequent cytosolic iron deposition following injury. Previous studies revealing iron presence in microglia have only considered this accumulation as iron retention and a signature of microglial activation . The idea of a link between iron management, by extent metabolism, and activated microglia has been contemplated for some time . Moreover, the capacity for RBC autophagy by microglial cells has already been well established as an important corrective response to CNS hemorrhage . In agreement with previous studies, we have observed microglial activation following FPI alone in the present work, as well as following chronic alcohol exposure [52, 53]. Interestingly, many of these activated microglia remained in the tissue at 1-day, only being present around RBCs and bleed sites 3-days post-FPI. These results demonstrate that morphology precedes function and, although reacting quickly to an injury, microglia may have a delayed immune response to invading RBCs. However, microglia may be responding to only atypical RBCs such as those dying from nutrient deprivation after being confined in tissue for 3-days post-FPI.
Other studies as well as our own also observed ethanol induced microglial activation [26, 54-56]. Expectedly, the combination likewise caused microglia activation and greater numbers to gather at sites with RBC aggregation (Fig. 7.B). Chronic alcohol use prior to FPI may be sensitizing microglia to stress and, in this manner, preconditioning the brain to future injuries. In other words, the CNS stays in ‘high alert’ for possible stress so that when stress does appear an appropriate response is rapidly mobilized. This state of hypervigilance may dysregulate inflammatory and immune responses to cause inappropriate and even exaggerated reactions. In this respect we observed multiple RBCs within activated microglia following EtOH + FPI. This excessive phagocytosis may either be due to chronic phagocytic activity or the engulfment of multiple RBCs at once. Similarly, the increased number of microglia present may either be the result of migration, division, or a degree of both. Our future work will include studies exploring these fundamental possibilities. A recent study has shown that microglia can assume a range of phenotypes under alcohol dependency and much remains to be understood in regard to their activation .
Collectively, these results begin to distinguish a pathway for iron maintenance by microglial cells. Activated microglia phagocytose and hemolyze RBCs, catabolize heme, and release free iron. Immediately, this iron gets bound to ferritin or hemosiderin. Any extracellular free iron becomes bound to apotransferrin or hemosiderin to form iron-containing complexes. Knowledge of macrophage iron management can be used to infer other details of the microglial-centric pathway. One role of macrophage is to phagocytose senescent RBCs . Therefore, in behaving like macrophage, activated microglia have been observed to also phagocytose RBC aggregations and, in so doing, regulate excess iron presence in the CNS. Heme transporters may also be involved in transporting heme into the cell or release it from plasma membrane-derived vacuoles following phagocytosis. Increases in the labile iron pool may cause excess iron to catalyze production of reactive oxygen species. Iron release may be inhibited by hepcidin hormone binding to and degrading ferroportin transporters. As iron can only bind when in the ferric form, membrane bound ferroxidases may be oxidizing the ferrous form. As an additional idea, the debris following RBC hemolysis and heme release may be shuffled to lysosomes for degradation (Fig. 8). Any lysosomal iron may be combined with partially degraded ferritin protein to create hemosiderin. Finally, additional stressors may free iron and evoke toxicity such as ferroptosis, an iron-dependent programmed cell death . Many directions remain to be studied in an effort to understand iron management by microglial cells. We have only begun to discover this alternative pathway of iron regulation.
4.2 Alcohol’s influence extends to iron management.
Being soluble in water, alcohol distributes into fluid spaces, as a result, higher concentrations of ethanol can be found in blood, or cerebral spinal fluid (CSF) surround the brain . As the major alcohol filtrating and metabolizing organ of the body, the liver becomes an obvious site for alcohol-induced iron build-up . In fact, this iron accumulation has been shown to contribute to the onset of alcoholic liver disease. This then develops into hepatitis and, over time, liver cirrhosis and destruction. In these same respects, generation of iron deposits in the brain may also lead to a form of inflammation and, if not corrected, neurodegeneration. We have shown that alcohol consumption increases bleeding and consequent iron deposition in the CNS (Fig. 7.B). In addition, alcohol abuse has also been shown to frequent rebleeding incidents and, in so doing, can augment deposition numbers even more . Despite this, we did not observe noticeable amounts of ferrous iron under EtOH + FPI. Therefore, although alcohol consumption significantly increases iron load in the CNS, the iron regulatory mechanisms adapt to accommodate this upsurge and prevent free iron accumulation and iron toxicity. To this effect, we have shown that prior chronic alcohol exposure changes the response of all three iron management pathways following FPI (Fig. 5). It may be that continued chronic alcohol consumption following FPI is required to release hemosiderin bound iron. This persistent stress would prevent inflammation from resolving and deregulate CNS functions. One affected system may be iron storage wherein the hemosiderin protein complex becomes destabilized and releases iron. Our future research will examine iron aggregation and regulation when chronic alcohol consumption is continued following FPI (an EtOH + FPI + ETOH model).