The lectin pathway (LP) of complement activation is believed to contribute to brain inflammation. The study aims to identify the key components of the LP contributing to TBI outcome as possible novel pharmacological targets. We compared the long-term neurological deficits and neuropathology of wild-type mice (WT) to that of mice carrying gene deletions of key LP components after experimental TBI. WT or MASP-2 (Masp2^−/−), ficolin-A (Fcna^−/−), CL-11 (Colec11^−/−), MASP-1/3 (Masp1^−/−), MBL-C (Mbl2^−/−), MBL-A (Mbl1^−/−) or MBL^−/− (Mbl1^−/−/Mbl2^−/−) deficient male C57BL/6J mice were used. Mice underwent sham surgery or TBI by controlled cortical impact. The sensorimotor response was evaluated by neuroscore and beam walk tests weekly for 4 weeks. To obtain a comparative analysis of the functional outcome each transgenic line was rated according to a health score calculated on sensorimotor performance. For selected genotypes, brains were harvested 6 weeks after injury for histopathological analysis. MASP-2^−/−, MBL^−/− and FCN-A^−/− mice had better outcome scores compared to WT. Of these, MASP-2^−/− mice had the best recovery after TBI, showing reduced sensorimotor deficits (by 33% at 3 weeks and by 36% at 4 weeks). They also showed higher neuronal density in the lesioned cortex with a 31.5% increase compared to WT. Measurement of LP functional activity in plasma from MASP-2^−/− mice revealed the absence of LP functional activity using a C4b deposition assay. The LP critically contributes to the post-traumatic inflammatory pathology following TBI with the highest degree of protection achieved through the absence of the LP key enzyme MASP-2, underlining a therapeutic utility of MASP-2 targeting in TBI.

Mannose-binding lectin (MBL), an initiator of the lectin pathway (LP) of complement activation, is detrimental in ischemic stroke, as shown in clinical studies and rodent models. Whereas humans have one functional MBL protein, rodents have two isoforms, MBL-A and MBL-C, whose functions relative to that of human MBL are unknown. To permit the clinical translation of preclinical data, we aimed to define the specific contributions of MBL-A and MBL-C to brain ischemia. We subjected mice with double (MBL^−/−) or single (MBL-A^−/− or MBL-C^−/−) MBL isoform depletion to transient middle cerebral artery occlusion (tMCAo). MBL^−/− mice had fewer neurological deficits and smaller ischemic lesions than WT mice. MBL-A^−/− mice had smaller lesions than WT mice and exhibited no significant behavioral defects, whereas MBL-C^−/− mice did not differ from WT mice. The induction of Mbl1 and Mbl2 (the MBL-A and MBL-C genes) expression 48 h after tMCAo was similar across genotypes. The time course of Mbl1 and Mbl2 expression in WT ischemic mice showed that Mbl1 activation occurred earlier (24 h) than Mbl2 activation (48 h). The plasma levels of MBL-A and MBL-C in MBL-C^−/− and MBL-A^−/− mice, respectively, were similar to those in WT mice both at baseline and at 48 h after tMCAo. At 48 h, MBL-A^−/− ischemic mice showed higher MBL-C levels in the brain than WT mice. WT and MBL-C^−/− ischemic mice had higher LP activity in plasma and, accordingly, higher levels of C3 deposition in the brain than MBL-A^−/− and MBL^−/− mice. In conclusion, mice with depletion of both MBL isoforms exhibited strong protection from ischemia/reperfusion injury. MBL-A was the main contributor to injury, likely owing to its earlier activation after ischemia and more efficient activation of the complement system than MBL-C.

The complement system is involved in promoting secondary injury after traumatic brain injury (TBI), but the roles of the classical and lectin pathways leading to complement activation need to be clarified. To this end, we aimed to determine the ability of the brain to activate the synthesis of classical and lectin pathway initiators in response to TBI and to examine their expression in primary microglial cell cultures. We have modeled TBI in mice by controlled cortical impact (CCI), a clinically relevant experimental model. Using Real-time quantitative polymerase chain reaction (RT-qPCR) we analyzed the expression of initiators of classical the complement component 1q, 1r and 1s (C1q, C1r, and C1s) and lectin (mannose binding lectin A, mannose binding lectin C, collectin 11, ficolin A, and ficolin B) complement pathways and other cellular markers in four brain areas (cortex, striatum, thalamus and hippocampus) of mice exposed to CCI from 24 h and up to 5 weeks. In all murine ipsilateral brain structures assessed, we detected long-lasting, time- and area-dependent significant increases in the mRNA levels of all classical (C1q, C1s, C1r) and some lectin (collectin 11, ficolin A, ficolin B) initiator molecules after TBI. In parallel, we observed significantly enhanced expression of cellular markers for neutrophils (Cd177), T cells (Cd8), astrocytes (glial fibrillary acidic protein—GFAP), microglia/macrophages (allograft inflammatory factor 1—IBA-1), and microglia (transmembrane protein 119—TMEM119); moreover, we detected astrocytes (GFAP) and microglia/macrophages (IBA-1) protein level strong upregulation in all analyzed brain areas. Further, the results obtained in primary microglial cell cultures suggested that these cells may be largely responsible for the biosynthesis of classical pathway initiators. However, microglia are unlikely to be responsible for the production of the lectin pathway initiators. Immunofluorescence analysis confirmed that at the site of brain injury, the C1q is localized in microglia/macrophages and neurons but not in astroglial cells. In sum, the brain strongly reacts to TBI by activating the local synthesis of classical and lectin complement pathway activators. Thus, the brain responds to TBI with a strong, widespread and persistent upregulation of complement components, the targeting of which may provide protection in TBI.

BACKGROUND: α-Syn (alpha-synuclein) increases and oligomerization contributes to ischemic brain damage. Nevertheless, the exact mechanisms of ischemia-induced α-Syn pathology after stroke are understudied. In this exploratory and hypothesis-testing study, we specifically investigated how ischemia-induced α-Syn pathology impacts vascular inflammation and angiogenesis. METHODS: Transient focal cerebral ischemia was induced in C57BL/6J wild-type and C57BL/6S mice with spontaneous deletion of α-Syn. In total, 72 mice were randomized to receive sham (12) or ischemia (60), with 1:1 allocation to genotype. Ischemic mice were included if presenting ≥3 intraischemic focal deficits. Experimental end points were 48 hours (acute phase) and 7 days (subacute phase) of reperfusion. Sensorimotor assessment, elevated plus maze, and survival analysis were stroke outcome readouts. Brain samples were collected for quantitative real-time polymerase chain reaction, Western blot, and immunohistochemistry. In vitro ischemia in brain microvascular endothelial cells was used to investigate the direct effect of extracellular α-Syn. RESULTS: In vivo, cerebral ischemia induced α-Syn 2.5-fold change gene expression, 2.7- to 3.2-fold-change protein oligomerization with apparent localization around cortical ischemic vessels. Improved subacute functional recovery linked to better survival was observed in C57BL/6S α-Syn null (87% to end point) versus C57BL/6J (45%) ischemic mice. The α-Syn null mice had reduced expression of ICAM-1 , MMP-9 , and vascular permeability factors, associated with less infiltrating leukocytes and juxta-vascular microglia in the acute phase. In the subacute phase, they showed upregulated proangiogenic factors ( VEGF-A , VEGFR-2 , Angpt-2 , and IL-6 ) and angiogenic vessels. In vitro, brain microvascular endothelial cells upregulated ICAM-1 , IL-6 , and VEGFR-2 expression when exposed to recombinant α-Syn at the beginning of ischemia and to a larger extent when α-Syn was preconditioned for 18 hours in hypoxia. CONCLUSIONS: These findings indicate that ischemia-triggered pathological α-Syn leads to worse outcome in stroke mice by promoting endothelial inflammatory response and immune cell infiltration during the acute phase and by limiting angiogenesis in the subacute phase.