We show here for the first time that TGFβ signaling in the brain increases in the first week after stroke in both young and old mice. We incorporated 18 month old mice into this study because nearly three quarters of all strokes occur in people over the age of 65 and there is a large knowledge gap regarding how mechanisms of recovery change with age. Similarly, we performed this study on mice of both genders in order to determine if there is a sex difference in TGFβ signaling after stroke. In 5 month old mice, TGFβ signaling increased 2 fold over baseline in the first week after stroke, before beginning to return to pre-stroke levels. This profile of increased TGFβ signaling was similar in 18 month old mice, although the absolute level of TGFβ signaling was significantly higher in the older animals. This could be in response to greater damage in the older animals as we found that lesion size was over double the volume compared to the younger mice. Baseline TGFβ signaling was also higher in the aged animals and so increased TGFβ signaling after stroke may also be a reflection of their higher baseline level. Our finding that infarct volume is increased in older animals conflicts with other studies that report that infarct volume is not increased in older subjects [18, 20]. However, these studies used the suture model of stroke, which causes a larger lesion than the dMCAO model of stroke used here. Studies that use a similar cortical model of stroke to the dMCAO model find that lesion size is increased in aged rats relative to young . Therefore the impact of age on lesion size appears to be stroke model dependent.
Every cell type in the brain has been shown to be capable of making TGF-β1 and increases in TGF-β1 mRNA have been demonstrated after stroke [1, 21]. To discover which cell type is predominantly responsible for post stroke production of TGF-β1 we co-localized an anti-TGF-β1 antibody with markers of different brain cell types. We found that TGF-β1 predominantly co-localized with CD68, a marker of activated microglia and macrophages, indicating that they are likely to be the cells that produce it after stroke.
While there is ample evidence that TGFβ production is increased after stroke, as it is after many kinds of brain injury, it was not known whether increased TGFβ production translates to an increased response, because all TGFβ isoforms are excreted as an inactive form that lies inert in the extracellular matrix . TGFβ receptor insertion into the membrane is highly regulated and the intracellular Smad pathways that transmit TGFβ signals interact with a complex array of other kinase substrates . Therefore, although many of the proteins that activate TGFβs are upregulated after brain injury, such as reactive oxygen species, metalloproteases, plasmin, and thrombospondin , it was not known if increases in TGF-β1 mRNA would correlate directly with increased TGFβ signaling after stroke. And in fact we found that TGFβ signaling was regulated differently in different cell types after stroke.
TGF-β1 is strongly neuroprotective, at least in part due to direct effects in neurons [11, 24–28]. It is also involved in synapse formation, the balance of excitatory and inhibitory transmission in the hippocampus and plasticity in multiple circuits . We found that TGFβ signaling in neurons is widespread and consistent before stroke and that there are no obvious quantitative or qualitative differences after stroke. This suggests that while TGFβ signaling plays an important constitutive function in neurons, the physiological role of increased TGF-β1 after stroke may not be to signal to neurons.
Surprisingly, we also found that the majority of oligodendrocytes in the brain are responding to TGFβ in the absence of injury and after stroke. This suggests that TGFβ signaling plays a physiological role in oligodendrocytes that was heretofore unappreciated. As with neurons, co-localization of pSmad2 with oligodendrocytes did not increase after stroke suggesting that the role of increased TGF-β1 after stroke is not to communicate to this cell type. Whether constitutive TGFβ signaling in oligodendrocytes is important for their function or survival remains to be elucidated.
We rarely found co-localization of pSmad2 with endothelial cells. Signal transduction by TGFβ family members is mediated via specific heteromeric complexes of type I and type II serine/threonine kinase receptors. In most cells TGFβ signals via the type I receptor ALK5 but in endothelial cells it can also signal via the type I receptor ALK1 [7, 30]. ALK5 induces phosphorylation of Smads 2 and 3 and ALK1 mediates phosphorylation of Smads 1, 5 and 8. Endothelial cells may not co-localize with pSmad2 due to the activation of the ALK1 pathway. Thus endothelial cells may still respond to TGFβ in the brain at baseline and after stroke but by activation of the Smads downstream of ALK1.
Our data demonstrates that CD68+ activated microglia and macrophages respond in an autocrine manner to the TGF-β1 they produce. We found that co-localization of CD68 with pSmad2 increased in parallel with TGFβ signaling reporter gene expression after stroke. TGFβ can exert either anti-inflammatory or pro-inflammatory effects in a context-dependent fashion. However, in the majority of contexts TGF-β1 appears to play an anti-inflammatory role and moderate the production of neurotoxic pro-inflammatory cytokines. It drives the differentiation of regulatory T cells and M2c macrophages that resolve immune responses, and resolves the inflammatory process in myocardial ischemia [7, 31, 32]. A function of TGF-β1 after stroke may be to drive the differentiation of monocytic lineage cells - both activated microglia and macrophages - into an M2c phenotype to enable a similar wound healing response to that identified in myocardial ischemia.
Our data also demonstrates that co-localization of pSmad2 with activated astrocytes increases after stroke. There is strong in vitro and in vivo evidence that TGFβ plays an important role in generating reactive astrogliosis, and that reactive astrocytes participate in innate immune responses. Overexpression of TGFβ chronically leads to reactive astrogliosis, and these mice display exacerbated reactive astrogliosis in a stab wound model . Injection of TGF-β1 also increases reactive astrogliosis after stab wound , while injection of neutralizing antibodies inhibits expression of fibrogenic proteins, including proteoglycans, and reduces reactive astrogliosis [5, 34]. Finally, Smad3 knockout mice display faster wound healing and decreased scar formation after brain stab injury . Prior to this study no investigation addressed whether it is TGFβ signaling in astrocytes that leads to reactive astrogliosis, but we show here that astrocytes do respond to post stroke increases in TGFβ. Together this supports the hypothesis that TGFβ signaling in astrocytes directly mediates astrogliosis after stroke. Astrogliosis may have a detrimental effect on recovery due to astrocytes extending processes in the infarct border to encompass the developing infarct, thereby inhibiting neuronal plasticity and the formation of new axons and blood vessels in the infarcted region .
As the brain ages it becomes characterized by a low grade chronic pro-inflammatory state and responds with an earlier and stronger innate immune response following stroke . We found that there is increased TGFβ signaling in the aged brain in the absence of injury and this may be part of an anti-inflammatory strategy employed by the aging brain to counter the primed pro-inflammatory state. Our findings are consistent with others who have shown that in aging the expression of TGFβ receptors increases, as does the expression of TGF-β1 [2, 20, 36, 37]. Conversely, since the data we present here strongly suggests that TGFβ signaling is important for neuronal function, increased TGFβ signaling in the aged brain may be required to maintain normal neuronal function in the face of age related changes. More research is needed to determine which of these scenarios is correct.