AT 1 inhibition mediated neuroprotection after experimental traumatic brain injury is dependent on neutrophils in male mice.

After traumatic brain injury (TBI) cerebral inflammation with invasion of neutrophils and lymphocytes is a crucial factor in the process of secondary brain damage. In TBI the intrinsic renin-angiotensin system is an important mediator of cerebral inflammation, as inhibition of the angiotensin II receptor type 1 (AT1) reduces secondary brain damage and the invasion of neutrophil granulocytes into injured cerebral tissue. The current study explored the involvement of immune cells in neuroprotection mediated by AT1 inhibition following experimental TBI. Four different cohorts of male mice were examined, investigating the effects of neutropenia (anti-Ly6G antibody mediated neutrophil depletion; C57BL/6), lymphopenia (RAG1 deficiency, RAG1-/-), and their combination with candesartan-mediated AT1 inhibition. The present results showed that reduction of neutrophils and lymphocytes, as well as AT1 inhibition in wild type and RAG1-/- mice, reduced brain damage and neuroinflammation after TBI. However, in neutropenic mice, candesartan did not have an effect. Interestingly, AT1 inhibition was found to be neuroprotective in RAG1-/- mice but not in neutropenic mice. The findings suggest that AT1 inhibition may exert neuroprotection by reducing the inflammation caused by neutrophils, ultimately leading to a decrease in their invasion into cerebral tissue.

Early DNase-I therapy delays secondary brain damage after traumatic brain injury in adult mice.

Traumatic brain injury (TBI) causes the release of danger-associated molecular patterns (DAMP) from damaged or dead cells, which contribute to secondary brain damage after TBI. Cell-free DNA (cfDNA) is a DAMP known to cause disruption of the blood-brain barrier (BBB), promote procoagulant processes, brain edema, and neuroinflammation. This study tested the hypothesis that administration of deoxyribonuclease-I (DNase-I) has a beneficial effect after TBI. Mice (n = 84) were subjected to controlled cortical impact (CCI) and posttraumatic intraperitoneal injections of low dose (LD) or high dose (HD) of DNase-I or vehicle solution at 30 min and 12 h after CCI. LD was most effective to reduce lesion volume (p = 0.003), brain water content (p < 0.0001) and to stabilize BBB integrity (p = 0.019) 1 day post-injury (dpi). At 6 h post injury LD-treated animals showed less cleavage of fibrin (p = 0.0014), and enhanced perfusion as assessed by micro-computer-tomography (p = 0.027). At 5 dpi the number of Iba1-positive cells (p = 0.037) were reduced, but the number of CD45-positive cells, motoric function and brain lesion volume was not different. Posttraumatic-treatment with DNase-I therefore stabilizes the BBB, reduces the formation of brain edema, immune response, and delays secondary brain damage. DNase-I might be a new approach to extend the treatment window after TBI.

AT2 activation does not influence brain damage in the early phase after experimental traumatic brain injury in male mice.

Antagonism of the angiotensin II type 1 receptor (AT1) improves neurological function and reduces brain damage after experimental traumatic brain injury (TBI), which may be partly a result of enhanced indirect angiotensin II type 2 receptor (AT2) stimulation. AT2 stimulation was demonstrated to be neuroprotective via anti-inflammatory, vasodilatory, and neuroregenerative mechanisms in experimental cerebral pathology models. We recently demonstrated an upregulation of AT2 after TBI suggesting a protective mechanism. The present study investigated the effect of post-traumatic (5 days after TBI) AT2 activation via high and low doses of a selective AT2 agonist, compound 21 (C21), compared to vehicle-treated controls. No differences in the extent of the TBI-induced lesions were found between both doses of C21 and the controls. We then tested AT2-knockdown animals for secondary brain damage after experimental TBI. Lesion volume and neurological outcomes in AT2-deficient mice were similar to those in wild-type control mice at both 24 h and 5 days post-trauma. Thus, in contrast to AT1 antagonism, AT2 modulation does not influence the initial pathophysiological mechanisms of TBI in the first 5 days after the insult, indicating that AT2 plays only a minor role in the early phase following trauma-induced brain damage.

Ribonuclease-1 treatment after traumatic brain injury preserves blood-brain barrier integrity and delays secondary brain damage in mice.

Traumatic brain injury (TBI) involves primary mechanical damage and delayed secondary damage caused by vascular dysfunction and neuroinflammation. Intracellular components released into the parenchyma and systemic circulation, termed danger-associated molecular patterns (DAMPs), are major drivers of vascular dysfunction and neuroinflammation. These DAMPs include cell-free RNAs (cfRNAs), which damage the blood-brain barrier (BBB), thereby promoting edema, procoagulatory processes, and infiltration of inflammatory cells. We tested the hypothesis that intraperitoneal injection of Ribonuclease-1 (RNase1, two doses of 20, 60, or 180 µg/kg) at 30 min and 12 h after controlled-cortical-impact (CCI) can reduce secondary lesion expansion compared to vehicle treatment 24 h and 120 h post-CCI. The lowest total dose (40 µg/kg) was most effective at reducing lesion volume (- 31% RNase 40 µg/kg vs. vehicle), brain water accumulation (- 5.5%), and loss of BBB integrity (- 21.6%) at 24 h post-CCI. RNase1 also reduced perilesional leukocyte recruitment (- 53.3%) and microglial activation (- 18.3%) at 120 h post-CCI, but there was no difference in lesion volume at this time and no functional benefit. Treatment with RNase1 in the early phase following TBI stabilizes the BBB and impedes leukocyte immigration, thereby suppressing neuroinflammation. RNase1-treatment may be a novel approach to delay brain injury to extend the window for treatment opportunities after TBI.

Correction to: Depletion of regulatory T cells increases T cell brain infiltration, reactive astrogliosis, and interferon-γ gene expression in acute experimental traumatic brain injury.

Following publication of the original article [1], the authors opted to correct the following mistakes. According to the title and our results, the conclusions in the abstract and at the end of the discussion the term "attenuates" must be corrected to read as "increases".

Depletion of regulatory T cells increases T cell brain infiltration, reactive astrogliosis, and interferon-γ gene expression in acute experimental traumatic brain injury.

BACKGROUND: Traumatic brain injury (TBI) is a major cause of death and disability. T cells were shown to infiltrate the brain during the first days after injury and to exacerbate tissue damage. The objective of this study was to investigate the hitherto unresolved role of immunosuppressive, regulatory T cells (Tregs) in experimental TBI. METHODS: "Depletion of regulatory T cell" (DEREG) and wild type (WT) C57Bl/6 mice, treated with diphtheria toxin (DTx) to deplete Tregs or to serve as control, were subjected to the controlled cortical impact (CCI) model of TBI. Neurological and motor deficits were examined until 5 days post-injury (dpi). At the 5 dpi endpoint, (immuno-) histological, protein, and gene expression analyses were carried out to evaluate the consequences of Tregs depletion. Comparison of parametric or non-parametric data between two groups was done using Student's t test or the Mann-Whitney U test. For multiple comparisons, p values were calculated by one-way or two-way ANOVA followed by specific post hoc tests. RESULTS: The overall neurological outcome at 5 dpi was not different between DEREG and WT mice but more severe motor deficits occurred transiently at 1 dpi in DEREG mice. DEREG and WT mice did not differ in the extent of brain damage, blood-brain barrier (BBB) disruption, or neuronal excitotoxicity, as examined by lesion volumetry, immunoglobulin G (IgG) extravasation, or calpain-generated αII-spectrin breakdown products (SBDPs), respectively. In contrast, increased protein levels of glial fibrillary acidic protein (GFAP) and GFAP+ astrocytes in the ipsilesional brain tissue indicated exaggerated reactive astrogliosis in DEREG mice. T cell counts following anti-CD3 immunohistochemistry and gene expression analyses of Cd247 (CD3 subunit zeta) and Cd8a (CD8a) further indicated an increased number of T cells infiltrating the brain injury sites of DEREG mice compared to WT. These changes coincided with increased gene expression of pro-inflammatory interferon-γ (Ifng) in DEREG mice compared to WT in the injured brain. CONCLUSIONS: The results show that the depletion of Tregs attenuates T cell brain infiltration, reactive astrogliosis, interferon-γ gene expression, and transiently motor deficits in murine acute traumatic brain injury.

Thrombin contributes to the injury development and neurological deficit after acute subdural hemorrhage in rats only in collaboration with additional blood-derived factors.

BACKGROUND: Acute subdural hemorrhage (ASDH) is a severe consequence of traumatic brain injury. The occurrence of subdural blood increases the lethality of these patients independent of the amount of blood or elevated intracranial pressure. Thrombin is one of the potential harmful blood components. Possible harmful effects of thrombin are mediated via the Protease-activated-receptor-1 (PAR1) and thus, translating the acute Thrombin release after ASDH into cell loss. The objectives of the present study were twofold, namely to examine (1) the impact of direct thrombin inhibition in the acute phase after hemorrhage on the long-term histological and functional deficits and (2) the early inhibition of PAR1 activation by thrombin with the selective antagonist SCH79797 on lesion volume at 14 days after ASDH. The effects of thrombin on the lesion size were investigated in two separate experiments via (1) direct thrombin inhibition in the subdural infused blood (Argatroban 600 µg) as well as by (2) intraventricular injection of the PAR-1 antagonist SCH79797 (1 µg or 5 µg). Lesion volume and behavior deficits using a neurological deficit score and a motor function test (beam balance test) were analyzed as outcome parameters at 14 days after injury. RESULTS: 59 Male Sprague-Dawley rats received a subdural infusion of 300 µl autologous blood or sham operation. Lesion volume at 14 days after ASDH tended to be smaller in the Argatroban-treated group when compared to the vehicle group (8.1 ± 1.1 vs. 10.1 ± 2.3 mm2, n.s.). Motor deficits in the beam balance test were not significantly less severe in the Argatroban-treated group. Animals treated with SCH79797 also showed a trend towards dose-dependent decreased lesion volume in comparison to the vehicle-treated group (1 µg: 4.3 ± 0.7 mm3; 5 µg: 3.8 ± 1.1 mm3; vehicle: 6.5 ± 2.0 mm3, n.s). CONCLUSIONS: Thrombin inhibition in the subdural blood and local cerebral blockade of PAR-1 cause a tendency towards reduced lesion volume or functional recovery. All results show a trend in favor of the acute treatment on the outcome parameters. Our results suggests that thrombin could be an important blood-derived factor during acute subdural hemorrhage that translates its deleterious effects in concert with other blood-induced factors.